title: 017 W vs H +- pABA

status: Public on May 31 2008

city: Ede

first: Arno

last: Wegkamp

address: Kernhemseweg 2

institute: NIZO food research

description: 017 W vs H +- pABA

hyb_protocol: Application This protocol can be used  for hybridisation of  Cy3/Cy5 labelled cDNA to  11K Agilent slides. It is an  abbreviated form of, and  an addition to the  manufacturer's protocol  (also available as pdf in  BASE under "protocols" as  "Agilent hyb. protocol v  4.1"). All chemicals  mentioned in the  hybridisation kit are  supplied in the Agilent in  situ hybridisation kit-plus  (Order# 5184-3568, lab  Arno, room temperature).  Micro array slides,  hybridisation chambers and  gasket slides (Order#  G2534-60002) are  available in Arno's lab. 20x  SSC (see NIZO QA  T/BFC063) is needed in  the wash solutions and has  to be made prior to the  washing steps. Hybridisation Tips. When the  volume of the required  amount of labelled cDNA  exceeds 40 µl evaporate  the excess of liquid in the  SpeedVac (reduction from  120 to 40 µl takes about  45 min without heating). Be very sure that  you make 210 µl of  hybridization mix, otherwise  the array chamber will not  be filled to at least 50%.  This will cause a "black  hole" in the centre of the  array image. In case you  conclude that you do not  have enough hybridization  mix you can add some  hybridization buffer (mix of  equal volumes water and  2x hybridization buffer).. Once the  chamber is closed, mark  the level of buffer on the  gasket slide (the one with  the rubber rings) using a  fine-liner. This allows you  to check afterwards  whether leakage has  taken place.. Cy-dye  bleaching has been shown  to be promoted by light,  ozone and humidity  (relevant for dried slides).  On hot summer days,  humid days or in the  vicinity of UV lamps dyes  may bleach very quickly. Hybridisation Special powder free gloves  are available for working  with slides. Avoid too much  light during the procedure  to prevent the cyanine  dyes from bleaching. 1.	Take the  necessary number of  Agilent slides from the  desiccator and close the  desiccator properly. Notate  the serial numbers in your  journal. The side containing the  bar code and "Agilent"  label contains the spots,  the side with the bar code  and the slide number label  is the back. The array that  is furthest away from the  label is array number 1, the  closest is array number 2  (see Figure 1). 2.	Prepare 0.3  g of  Cy3 and 0.3  g of Cy5  labelled cDNA's as  described in "cDNA  synthesis and labelling"  each in a volume of 40 µl  (see tips if concentration is  too low, or add water if  concentration is too high.  Also see note below) 3.	Mix the Cy3 and  Cy5 labelled cDNA's in an  amber Eppendorf tube 4.	Heat at 98 °C for  3 minutes and cool down  to room temperature (use  clamp or pierce a hole in  the lid to avoid  spontaneous opening of  the lids when heating) 5.	Add 25 µl of 10x  control (freezer, Arno) 6.	Add 105 µl 2x  hybridisation buffer  (hybridisation kit) The total volume will now  be 210 µl 7.	Mix (no vortex to  avoid bubble formation)  and spin briefly to collect  all of the mix at the bottom  of the tube 8.	Put a gasket  slide (G2534-60002) in the  microarray hybridisation  chamber (G2534A) and  pipet the hybridisation  mixtures in the centre of  the array frames without  introduction of bubbles 9.	Put the  microarray slide slightly on  top of it without  introduction of bubbles (the  side containing the bar  code and "Agilent" label  has to face towards the  hybridation mixture). The  easiest way to achieve this  is to put one short side of  the microarray slide on the  gasket slide and slowly  lower the array slide 10.	Place the upper  part of the hybridisation  chamber (chamber cover)  and the chamber clamp  and close this by turning  the thumbscrew clockwise  and tighten it firmly by hand  (see Figure 2) Do not use tools to tighten  the thumbscrew when  closing the hybridisation  chamber!! 11.	Take the  hybridisation chamber and  turn it vertically to check  for correct bubble  formation. Bubbles are  correct when they move  freely in the hybridisation  chamber and the mixture  can reach the complete  surface of the slide upon  turning of the slide. Also  check whether the  chamber is filled to at least  60% of its total volume 12.	Hybridise for 17  hours at 60 °C. If  necessary balance the  rotor with empty  hybridization chambers Note about the amount of  cDNA to be used per array We are still discussing  whether the amount of  cDNA to be used on a  microarray should be  normalized with respect to  the concentration of cDNA  measured in the sample or  with respect to the  concentration of Cy-dye  measured. Amersham  recommends the second  option. However, until now  we have used the first  option (0.3  g cDNA per  array). Until now we have  observed ratios of  Cy-dye/cDNA of 0.03 to  0.12 pmol/ng yielding good  final results.   Washing Tips. Before starting  the washing steps prepare  all washing solutions and  put them in washing trays  (lab Arno). If more than one  slide has to be washed,  separate all array and  gasket slides as described  and keep the array slides  submerged in solution 1  until all slide sandwiches  are separated. Continue  with the washing steps for  all slides at the same time.. To avoid  exposure to light place a  box over the washing trays  when washing the slides. Preparation of wash  solutions Wash solution 1 (6x SSC,  0.005% Triton X-102) 700 ml	milliQ 300 ml	20x SSC  (NIZO-SOP T/BFC063) 0.500 ml	10% Triton  X-102 (hybridization kit). Mix the  components and pass the  solution through a 0.22 µm  filter. Store at room  temperature  Wash solution 2 (0.1x  SSC, 0.005% Triton X-102) 995 ml	milliQ 5.0 ml	20x SSC  (NIZO-SOP T/BFC063) 0.500 ml	10% Triton  X-102 (hybridization kit). Mix the  components and pass the  solution through a 0.22 µm  filter. Store at 4 ºC Washing 1.	After  hybridisation remove the  glass slide from the  hybridisation chamber and  separate the glass slides  completely submerged in  wash tray 1 containing  wash solution 1 at room  temperature by inserting  the blunt tip of the plastic  tweezers at the bar code  end of the slide sandwich  and gently turning the tip of  the tweezers. 2.	Transfer the  slides to wash tray 2  containing wash solution 1  and a magnetic stirrer and  place this on a magnetic  stir plate. Wash the slides  for 10 minutes at room  temperature at medium  stirring. 3.	Transfer the  slides to wash tray 3  containing pre-cooled  wash solution 2 and a  magnetic stirrer and place  it in another tray filled with  melting ice on a magnetic  stir plate. Wash for 5  minutes at medium stirring. 4.	Dry the slides by  blowing all droplets of  washing solution to the  edges of the slides using a  nitrogen airgun (Arno).  Adjust the manometer to 1  atm. 5.	Put the slides in  a dark dust-free box  (microscope slide box, at  the reader) and scan the  slide immediately (see  protocol "Scanning of  DNA microarrays").    Figure 1. Our convention  for numbering the arrays on  an Agilent slide.   Figure 2. Turning the  thumbscrew hand-tight.

scan_protocol: Application This procedure can be  followed to scan DNA  micro array slides on the  Scan Array Express  microarray scanner. Operating the array  scanner 1.	Turn on the  scanner and the computer  (in this order) 2.	Login: scanner,  password: scanner2 3.	double click the  'ScanArray Express' icon 4.	Switch on lasers  1 and 3 at least 15 minutes  prior to scanning arrays 5.	Put focus on file This is to prevent you from  accidentally turning of the  laser again after the 15  minutes warming-up phase 6.	Press 'Scan |  Prescan' 7.	Put resolution at  50 nm, and PMT value for  both channels at for  instance 70% (spotted  arrays) or 40% (Agilent  arrays) 8.	Press 'Start' 9.	Press 'Palette,'  'Green' as soon as  projection of the image has  started (first dye), select  the red colour as soon as  scanning of the second  layer (second dye) has  started 10.	Select frame for  high resolution scan, adjust  resolution (to 10 nm) and  scan the array for both  dyes. Adjust PMT values  to balance signals  obtained for both channels. Balance both  channels as well as  possible to achieve a  comparable average  intensity for both channels.  This avoids large  normalisation corrections  during analysis of the data,  so decreases the chance  on introduction of  artefacts.. Make at least  one scan in which none of  the spots are saturated.  Run this scan at for  instance PMT = 40% and  step-wise increase the  PMT level until both  channels have comparable  intensities (e.g. 1000  intensity units) 11.	Press 'File |  Save' to save the files,  'Save all' saves both dye  layers. Create a new  folder for each array. 'Save all' saves  the signals from both layers  in individual files. It's possible to  save the 'composite' file in  .jpg format, however this is  not necessary since al  data are in the original files  as well 12.	Switch off the  lasers and the scanner  after use Tips. Allow the lasers  to warm for at least 15  minutes   Quantification of spots 1.	Start Imagene by  double clicking the  'Imagene' icon 2.	Load tiff files of  both channels 3.	Load the  template file from the folder  'GAL files' located on the  desktop Every batch of arrays has  it's own GAL file. The  name of the GAL file  corresponding to the batch  of slides can be found  under 'Array LIMS | Array  designs' as 'print  map/reporter map' in BASE  (http://kluyver/base). (e.g.  LpGn07.gal for the several  batches of spotted L.  plantarum arrays from  Groningen and  '012731_D_20040830.gal'  that was edited to  lpag01.gal for the 1st  design of Agilent L.  plantarum slides) 4.	Adjust frame by  pressing the 'Rectangle  adjust' button, drag the  circles on the spots,  starting with the left-up  spot (this is not necessary  for Agilent slides since they  are recognised  automatically) 5.	Press the  'Automatically place grid'  button and check by eye if  the grid was placed  correctly 6.	Press 'Quantify'  (a table with spot id's and  intensities is produced) 7.	Save files (for  both dyes) in the same  directory as the original tiff  files are located just by  selecting (single click) the  directory (don't open!) and  press the 'Save' button 8.	Remove slides  by pressing 'File,' 'Remove  selected images' for both  channels before loading  and quantification of other  arrays (only necessary  when more slides are to be  quantified) Repeat steps 14, 16-20 to  load and quantify multiple  slides after the current  slides have been removed.  Repeat step 15 when  another type/batch of  slides is used. 9.	Import data in  BASE  (http://kluyver/base)   Although the choice is  completely arbitrary, for  clarity always use channel  1 for the Cy3 label and  channel 2 for the Cy5 label  to avoid accidental  swapping of labels during  data analysis.

data_processing: After blank spots had been  removed, array  measurements were  normalized by local fitting  of an M-A plot applying the  loess algorithm, using the  Limma package in R  (http://www.r-project.org).  Spot intensities in both  channels (I1 and I2) were  thus converted to M-A  coordinates, where M =  log2(I1/I2) and A =  log2(I1/I2)/2. Normalized  intensities were used for  further analysis.   The statistical significance  of differences was  calculated from variation in  biological replicated, using  the eBayes function in  Limma (cross-probe  variance estimation) and  Holmes determination of  significance. Only genes  with a log2 ratio of -1 and  +1 and a Holmes value  less than 0.1 were  considered significant.   The batch experiment with  and without pABA scheme  consisted of a loop design  with 21 microarrays with  the following samples  hybridized on one array  and labeled with Cy3 and  Cy5, respectively: C1+P  and F1+P, F1+P and  C2+P, C2+P and F3+P,  F3+P and C3+P, C3+P and  F2+P, F2+P and C1+P,  C1+P and C2+P, F2+P and  F3+P, C1-P and F1-P, F1-P  and C2-P, C2-P and F3-P,  F3-P and C3-P, C3-P and  F2-P, F2-P and C1-P, C1-P  and C2-P, F2-P and F3-P,  C3-P and F1+P, F2+P and  C1-P, C2+P and F3-P,  F1-P and C3+P, and F2+P  and F1-P. Here, C1+P,  C2+P, C3+P, F1+P, F2+P,  and F3+P represent  biological triplicates of the  L. plantarum harboring  pNZ7021 and pNZ7026,  respectively, when grown  in batch in the presence of  pABA. The C1-P, C2-P,  C3-P, F1-P, F2-P, and  F3-P, represents the L.  plantarum harboring  pNZ7021 and pNZ7026,  respectively, when grown  in batch in the absence of  pABA.

organism: Lactiplantibacillus plantarum

source_name: Lactobacillus plantarum pNZ7021 (3)

label_protocol: General comments Tips. Be sure that  your RNA is of good  quality before you start.. Use PCR  machine 3 or 4 (suitable  for 0.2 ml tubes) for  incubation steps,  (program 70marc and  42marc).. Place all the  reagents (except for the  RT enzyme) on ice to  thaw.. Chemicals  descriptions can be  found on page 6.. All reagents  are in room A128. If you  take a new batch,  please inform beheerder  (= Marc Stevens).. This protocol  is made for the so-called  "Home-made kit". The  protocol for the  Amersham post-labelling  kit is the same, except  for the chemical's name.  Please follow the  manufacturer's protocol  when in doubt.. A micro-array  file can be found in  room A128 with more  information, order  numbers and  manufacturer's  protocols.. While  purifying the labelled  cDNA, the column  should still be coloured  even after tree wash  steps, when you have  labelled total RNA from  bacteria. If you do not  see the dye colours at  this stage, you will  probably not have  enough labelled cDNA  to obtain good  hybridization results.. Labelled  cDNA can be stored at  -20°C, but only for a  short period. In  particular the Cy5 label  quickly bleaches. Do  not use labelled DNA  older than a few days,  or you may get very low  signal on the array.. Cy-dye  bleaching has been  shown to be promoted  by light, ozone and  humidity. On hot summer  days, or in the vicinity of  UV lamps dyes may  bleach very quickly.. Prepared  Cy-dyes can be stored  up to tree months at  -80°C.. Note that the  relation between the  amount of RNA that  goes into the reaction  and the amount of  cDNA synthesized is not  proportional (see figure  below by Roger  Bongers)   Things that can go  wrong (and already  went wrong once). Wash with  EtOH (80%), elute with  NaCO3 during the first  purification.. Work in the  dark from the moment  you see colours in your  sample.. Add 40 ml of  100% EtOH to the wash  buffer of the GFX  column purification kit.   cDNA Synthesis General comments. Nuclease-free  water is available in  PCR kits or can be  ordered from commercial  suppliers.. It is not useful  to add more than 25 µg  RNA to the Annealing  Mix, because this will  not lead to higher cDNA  yields (see figure on  page 1). Annealing Annealing Mix x µl	RNA  preparation (25 µg RNA  for spotted arrays or 5 to  10 µg RNA for Agilent  arrays, see tips) y µl	nuclease-free  water 1 µl	Random  Nonamers 11 µl	TOTAL  1.	Mix Annealing  Mix in a 200 µl PCR  tube gently by pipetting  up and down 2.	Incubate for 5  min. 70°C 3.	Cool at room  temperature for 10 min.  (annealing) 4.	Spin down  the mixture to the  bottom of the tube 5.	Place on ice Reverse transcription Reverse Transcription  Mix 4  l	5x buffer 2  l	0.1 M DTT 2  l	dNTP-mix  (with AA-dUTP [home  labelling kit] or 1 + 1  l  for Amersham kit, see  Manufacturers Protocol) 1  l	TMIII (keep  only briefly outside   20°C) 20  l	TOTAL  1.	Mix by very  gently by pipetting up  and down (vigorous  pipetting will denature  the enzyme!) Before pipetting the  enzyme: spin down the  residue in the tube to be  sure that all enzyme is  finished before starting a  new kit! 2.	Incubate for 3  hrs at 42°C (in  PCR-machine) 3.	Cool on ice  for immediate  purification or place at   20°C for storage Degradation of mRNA 1.	Add 2  l 2,5 M  NaOH 2.	Mix (vortex)  and spin down 3.	Incubate for  15 min. at 37°C 4.	Add 10  l 2 M  HEPES free acid 5.	Mix (vortex)  and spin down 6.	Ready for  purification or store at   20°C   Labelling of cDNA with  cyanine dyes General comments. Use 80%  ethanol as wash buffer.. The small  amount of Tris present  in the kit wash buffer  can react with the  Cy-dye NHS Esters,  reducing the yield of  labelling during the  coupling step.. Prepare fresh  Sodium Bicarbonate 0.1  M, pH 9.0 (see page 6)  immediately prior to use  in a labelling reaction, or  use an aliquot from a   20°C stock (do not  freeze it again!). Purification of amino  allyl-modified cDNA 1.	For every  cDNA reaction to be  purified, place one  CyScribe GFX column  into a clean collection  tube. Add 500  l of  capture buffer to each  CyScribe GFX column 2.	Transfer the  unpurified cDNA  products into each  CyScribe GFX column;  mix the cDNA by gently  pipetting up and down 5  times 3.	Centrifuge  each column in a micro  centrifuge at 13.800 x g  for 30 seconds 4.	Remove the  CyScibe GFX column  and discard the liquid at  the bottom of each  collection tube. Return  each CyScribe GFX  column into the used  collection tube 5.	Add 600  l of  80% ethanol to each  column and centrifuge  at 13.800 x g for 30  seconds 6.	Remove the  CyScibe GFX column  and discard the liquid at  the bottom of each  collection tube. Repeat  the wash step twice for  a total of three washes.  After the final wash,  discard the liquid and  place the column back  in the used collection  tube 7.	Centrifuge  each column in a  microcentrifuge at  13.800 x g for an  additional 10 seconds to  remove all wash buffer  in the tip of the column.  Discard the collection  tube 8.	Transfer each  CyScribe GFX column to  a fresh 1.5 ml micro  centrifuge tube and add  60 µl of fresh 0.1 M  sodium bicarbonate  pH9.0 directly to the top  of glass fiber matrix in  each CyScribe GFX  column. It is crucial that  the elution buffer  completely covers the  membrane 9.	Incubate the  CyScribe GFX column at  room temperature for 1-5  minutes. Centrifuge at  13.800 x g for 1 minute  to collect the purified  labelled cDNA 10.	Repeat this  elution step one more  time 11.	Proceed  immediately to the  labelling reaction Labelling of amino  allyl-modified cDNA with  Cy-dye 1.	Add the  amino allyl modified  cDNA (in 0.1 M sodium  bicarbonate) directly into  one aliquot of CyDye  NHS Ester. Resuspend  the ester completely by  pipetting several times  and transfer the solution  to an amber 1.5 ml tube 2.	Incubate at  room temperature, in the  dark for 60 to 90  minutes 3.	Add 15 µl 4 M  Hydroxylamine to each  coupling reaction 4.	Mix by stirring  and incubate at room  temperature, in the dark,  for 15 minutes 5.	Proceed  directly to purification of  Cy-dye labelled cDNA Purification of Cy-dye  labelled cDNA 1.	For every  cDNA reaction to be  purified, place one  CyScribe GFX column  into a clean collection  tube. Add 500 µl of  capture buffer to each  CyScribe GFX column 2.	Transfer the  unpurified cDNA  products into each  CyScribe GFX column;  mix the cDNA by gently  pipetting up and down 5  times 3.	Centrifuge  each column in a micro  centrifuge at 13.800 x g  for 30 seconds 4.	Remove the  CyScibe GFX column  and discard the liquid at  the bottom of each  collection tube. Return  each CyScribe GFX  column into the used  collection tube 5.	Add 600 µl of  wash buffer (from the  kit) to each column and  centrifuge at 13.800 x g  for 30 seconds 6.	Remove the  CyScibe GFX column  and discard the liquid at  the bottom of each  collection tube. Repeat  the wash step twice for  a total of three washes.  After the final wash,  discard the liquid and  place the column back  in the used collection  tube 7.	Centrifuge  each column in a  microcentrifuge at  13.800 x g for an  additional 10 seconds to  remove all wash buffer  in the tip of the column.  Discard the collection  tube 8.	Transfer each  CyScribe GFX column to  a fresh 1.5 ml amber  micro centrifuge tube  and add 60 µl of elution  buffer (from the kit)  directly to the top of the  glass fiber matrix in each  CyScribe GFX column. It  is crucial that the elution  buffer completely covers  the membrane CyDye-labelled probes  bind tightly to GFX resin.  Never use water to  elute these labelled  probes because probe  yield will be inconsistent  and lower than that  obtained with elution  buffer Pre-warming elution  buffer to 65°C might  increase the labelled  cDNA yield by  approximately 5% 9.	Incubate the  CyScribe GFX column at  room temperature for 1-5  minutes. Centrifuge at  13.800 x g for 1 minute  to collect the purified  labelled cDNA 10.	Repeat this  elution step one more  time 11.	Measure the  concentration of cDNA,  and the amount of  incorporated Cy-dye  using the "microarray"  program of the  nanodrop (see note  below). 12.	Ready for  hybridization or store at   20°C Notes about labelled  cDNA absorption  spectrum. The 260/280  nm absorption ratio  should be in the range  1.6 to 1.8. The spectrum  of the incorporated  Cy-dye as recorded with  the nanodrop gives you  important information  about the fluorescence  processes going on. In  particular, a high ratio of  the first absorption peak  relative to the second  will indicate high  self-quenching due to  overloading of the  cDNA with dye. If the  peaks are almost  equally high then the  labelling density is too  high and fluorescence  will be low due to  self-quenching.   Home-made labelling kit  components. TMIII =  Superscript TMIII  (Invitrogen, order #  18080-044). DTT is  supplied with TMIII  enzyme. 5 x Buffer is  supplied with TMIII  enzyme. GFX Columns  can be ordered from  Amersham Bioscience,  order # 27-9606-02. NaOH,  HEPES, Hydroxylamine,  and Sodium  Bicarbonate solutions  should be made by the  researcher.. dNTP's can  be ordered from  Amersham Bioscience,  order # 27-2035-01. The random  nonamer stock (to be  diluted 1/20) can be  found in room A128  Hydroxylamine 4 M	 Hydroxylamine Prepare freshly  Sodium Bicarbonate 0.1  M, pH 9.0 0.84 g	NaHCO3 90 ml	Milli-Q water. Adjust to  exactly pH 9.0 with 1 M  NaOH. Adjust volume  to 100 ml with Milli-Q  water. Store in  aliquots or prepare  freshly. Do not freeze  again after use  Random Nonamers 2 µl	random  nonamer stock (27.1  µg/µl) 38 µl 	nuclease-free  water  AA-dUTP 1 mg	AA-dUTP  (Sigma, order # A0410) 20 µl	K-phosphate,  0.1M, pH 7.5  dNTP-mix 5 µl	100 mM of  each dATP, dCTP and  dGTP 3 µl	100 mM  dTTP 2 µl	AA-dUTP-mix Add 40 µl	Water  Cy5-dye or Cy3-dye  NHS Ester 1 tube	Cy5 or Cy3  monofunctional reactive  dye (Amersham, order #  PA23001) 40 µl 	DMSO Store in 8 x 5 µl aliquots  at  80°C up to 3 months Use 5 µl per labelling   Toxic chemicals.  HYDROXYLAMINE  (See Chemical safety  cards on  http://kluyver/intranet/). Chemical  dangers: May explode  on heating above 70°C  or when exposed to  open flame. The  substance decomposes  rapidly at room  temperature, especially  in presence of moisture  and carbon dioxide, and  violently on heating  producing toxic fumes  including nitrogen  oxides. The solution in  water is a weak base.  Reacts violently with  oxidants, metals such as  finely divided zinc, some  metal oxides,  copper(II)sulfate and  phosphorus chlorides  causing fire and  explosion hazard.. Effects of  short-term exposure:  The substance irritates  the skin and the  respiratory tract, and is  corrosive to the eyes.  The substance may  cause effects on the  blood, resulting in  formation of  methaemoglobin. The  effects may be delayed.  Medical observation is  indicated.. Effects of  long-term or repeated  exposure: Repeated or  prolonged contact may  cause skin sensitization.  The substance may  have effects on the  blood, resulting in  formation of  methaemoglobin and  the consequent  anaemia.

extract_protocol: Isolation of RNA General comments. Both  Biophenol and Acidic  phenol (Sigma) are  suitable for application  in the Extraction  Mixture.. The ratio of  pellet to Extraction  Mixture seems to affect  the RNA amounts and  quality. 50 ml MRS  culture of an OD600 of  1 gives reproducibly  good results. Five times  this amount, however,  yielded poor quality  RNA (Personal  communication from  Arno Wegkamp and  Mariela Hebben).. The  quenching buffer  contains methanol.  Supernatant from the  quenching procedure  should be discarded in  the appropriate  container.. The  extraction buffer  contains phenol. Phenol  is a toxic compound and  waste containing phenol  should be discarded in  the appropriate  containers. Collecting cells Quenching Buffer 66.7 mM	HEPES (pH  6.5) 60%	Methanol  Extraction Mixture 500 µl	 Phenol/Chloroform 30 µl	10% SDS 30 µl	3 M NaAc  (pH 5.2) 500 mg	glass-beads  (75-150 µm) 400 µl	TE buffer (or  MRS) Prepare in a screw-cap  tube  1.	For each  sample prepare a tube  with Extraction Mixture Make sure these  components are mixed  freshly each time, so do  not freeze tubes  containing Extraction  Mixture. 2.	Harvest cells  and quench them  immediately in  Quenching Buffer. Add  4 volumes of Quenching  Buffer ( 40°C) to 1  volume of cell culture 3.	Mix and store  the sample at -40°C 4.	Centrifuge  cells at  20 C at 9000  rpm for 10 minutes  (Centrifuge RC5B, room  A156) 5.	Discard the  supernatant; keep the  cell pellet cooled in  ethylene glycol (60 %)  and work quickly to  prevent condensation of  water. Transfer the  pellet with a pre-cooled  spatula to a screw-crap  tube with Extraction  Mixture The supernatant  contains methanol and  should be discarded in  the appropriate  container 6.	Mix the cell  pellet thoroughly with  Extraction Mixture by  shaking by hand 7.	Freeze the  tube immediately in  liquid nitrogen 8.	Store at  80°C  or isolate RNA  immediately RNA isolation Tips:. Work as  quickly as possible and  keep the samples cool.. Do not break  too much samples at  once (max 4) to enable  you to work quickly.. Break the  cells immediately in  frozen condition. Do not  thaw the samples from   80°C. . Keep your  samples cool during  processing.. Use filter tips  to avoid contamination  from pipets.. Wear gloves  and lab coat.. For more tips  look at the Ambion  website:  http://www.ambion.com The following steps  should be performed as  swiftly as possible  before cDNA labelling. 1.	Break cells in  the Savant FastPrep  FP120. Three times 40  seconds at speed 4.0  (cool cells between  steps if necessary) 2.	Centrifuge for  1 minute at 4°C in an  Eppendorf centrifuge at  20.800 g. 3.	Transfer 500  µl of the supernatant to  a new tube. Add 400 µl  chloroform (chloroform  should be cooled at  4°C) 4.	Centrifuge for  1 minute in an  Eppendorf centrifuge at  full speed (4°C). 5.	Take the  supernatant and mix it in  1:1 ratio with the  capture buffer of the  RNA-isolation kit. 6.	Continue with  the "High Pure RNA  Isolation Kit" from  Roche (order # 1 828  665) 7.	Incubate for  60 minutes with DnaseI,  elute with 50 µl elution  buffer 8.	Store the  RNA in at least 2  aliquots, one of 20 µl  (for concentration and  quality analysis and  labelling) and one  back-up of 30 µl at   80°C. RNA quality control Measuring the RNA  concentration . Measure the  concentration of RNA at  260 nm by dissolving 1  µl of a preparation in  500 µl of water.. 1 absorption  unit at 260 nm is  equivalent to 40 µg  RNA/ml.. The  A260/A280 ratio should  be in the range of  1.9-2.0. The closer to  2.0, the more pure the  RNA will be. When the  ratio is lower than 1.9,  DNA is still present.  When lower than 1.8,  proteins are still present.. The  recommended RNA  concentration for  labelling is 20-25 µg /µl .. The minimal  RNA concentration  should be 1 µg/µl. Quality check on the  Bioanalyzer. First  determine the  RNA-concentration  using the "nanodrop".. The  Bioanalyzer can be  found in room A230.. For the quality  check you have to use  the Nano-assay chips.. Try to apply  12 samples to the  Bioanalyzer, but, on the  other hand, do not wait  for a week to analyse  your samples.. Please follow  the instructions of the  manufacturer or ask the  instrument custodian (=  Anne Wiersma).. The ratio  between the 23S and  the 16S RNA should be  at least 1.6. Lower ratios  are an indication of  extensive RNA  degradation.. The  "zero-line" in the  electrogram should be  more or less straight  (when in doubt, ask an  experienced  researcher).. The analysis  on the Bioanalyzer is a  qualitative assay, not a  quantitative assay. The  concentrations found in  the spectrophotometer  are more reliable.

tag: Lactobacillus plantarum pNZ7021 (3) - pABA Cy3

source_name: Lactobacillus plantarum pNZ7026 (1)

tag: Lactobacillus plantarum pNZ7026 (1) + pABA Cy5


title: 019 W vs H +- pABA

type: RNA

country: Netherlands

description: 019 W vs H +- pABA

molecule: total RNA

source_name: Lactobacillus plantarum pNZ7021 (2)

tag: Lactobacillus plantarum pNZ7021 (2) + pABA Cy3

source_name: Lactobacillus plantarum pNZ7026 (3)

tag: Lactobacillus plantarum pNZ7026 (3) - pABA Cy5


title: 014 W vs H - pABA

description: 014 W vs H - pABA

source_name: Lactobacillus plantarum pNZ7026 (2)

tag: Lactobacillus plantarum pNZ7026 (2) - pABA Cy3

source_name: Lactobacillus plantarum pNZ7021 (1)

tag: Lactobacillus plantarum pNZ7021 (1) - pABA Cy5


title: etiolated, dark  - SlideA - rep 4

status: Public on Jun 01 2008

city: Stanford

first: John

middle: F

last: Fernandes

state: CA

address: 385 Serra Mall

department: Bio Sci

country: USA

institute: Stanford University

description: total RNA extracted from pools of 10 siblings

hyb_protocol: Arrays were hybridized for 14 hours at 42 C in a hybridization buffer containing 50% formamide, 5X SSC, and 0.1% SDS.  After hybridization, arrays were initially washed at 42 C in 2X SSC, 01% SDS for 5 minutes and then subject to two, five minute washes in 0.1X SSC at room temperature.  Complete details are available at http://www.maizearray.org/files/Hybridization_Protocol_For_cRNA_Targets.pdf

scan_protocol: Arrays were scanned on an Axon 4100 AL scanner (Molecular Devices Corporation, Sunnyvale CA) at 95% laser power.  Raw data was extracted from the resulting images using the GenePix 6.0 software. (Molecular Devices Corporation, Sunnyvale CA)

data_processing: Each sample was hybridized 4 times to different other samples. The 2 dye intensities were imported into R and normalized with limma ( (lowess for within array, quantile for between arrays). Then the individual intensities were calculated and the 4 measurements for each sample were averaged..

organism: Zea mays

source_name: etiolated, dark seedling

label_protocol: Total RNA (~1.5 ug) was labeled with either Cy3 or Cy5 using the Ambion Message Amp II target labeling kit (Ambion Cat # 1751) according to the manufacturers recommendations using reduced reaction reagents and volumes in order to conserve reagents.  Complete details are available at http://www.maizearray.org/files/cRNA_Target_Production_Using_RNA_Amplification.pdf

growth_protocol: planted 2 cm deep in 5 cm of sterilized vermiculite in plastic trays with bottom drainage holes to prevent water-logging.  Trays were saturated with distilled, deionized water then placed under fluorescent illumination (16 h 350 uE m-2) on racks in a walk-in 26ºC room.

extract_protocol: Trizol reagent (Invitrogen), purified by ethanol precipitation.  Samples were concentrated using Qiagen (Valencia CA) MinElute columns

treatment_protocol: tray covered with 3 layers of heavy duty aluminum foil for 7 days, watered only on days 3 and 6

tag: Strain

value: B73, Tissue:whole seedling, Age:7 days

tag: Treatment


title: 013 W vs H - pABA

description: 013 W vs H - pABA

tag: Lactobacillus plantarum pNZ7026 (2) - pABA Cy5


title: Biological Replicate 1 Technical Replicate 3 HOG1-GFP time 60

city: Berkeley

first: Jesse

middle: C

last: Patterson

address: 528 Barker Hall

department: Dept of Molecular and Cell Biology

institute: UC-Berkeley

description: Arrays were post proccessed according to the protocol found at: http://cat.ucsf.edu/pdfs/hybCourseProtocols_v2.6.pdf

hyb_protocol: Sample 	             7.5 ul    (2.5 ug), Pool 		7.5 ul  (2.5 ug), 20X SSC	             3 ul, PolyAdenlylic acid	1.5 ul, 1M HEPES	0.5 ul, 10% SDS	             0.5 ul. boil for 1:30 mins, let cool to room temperature. Apply to arrays, in metal hybridization chambers, hyb at 63C for 24 hours.

scan_protocol: Scanned with Axon GenePix 4000B scanner.

data_processing: Unnormalized GPR files from GenePix were converted to .mev files by TM4 express converter.  .mev files were then normalized by TM4 MIDAS v2.20 beta using LOWESS and SD regularization algorithms.  Values correspond to the log2 ratios of the normalized data.  All data analysis was done using TIGR TM4 MeV v4.0.01.  The un-normalized .gpr files are attached as supplementary files.

organism: Saccharomyces cerevisiae

source_name: Reference pool

label_protocol: 2.5 ug aRNA in 0.1 M Sodium Bicarbonate and 50% DMSO, clean in Zymo Research RNA clean up columns.

extract_protocol: Total RNA extracted according to the Ambion RiboPure(TM)-Yeast kit protocol.  1 ug total RNA was then used to amplify amino-allyl UTP antisense RNA accoding to the Ambion Amino Allyl Message AMP(TM) II aRNA Amplification Kit protocol.

tag: Pooled aRNA from all 6 samples in technical replicate 3

source_name: B1 T3 HOG1-GFP 60 mins

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-GFP strain at 60 mins hyperosomotic shock with 1 M sorbitol


title: Biological Replicate 1 Technical Replicate 1 HOG1-GFP time 60

tag: Pooled aRNA from all 6 samples in technical replicate 1

source_name: B1 T1 HOG1-GFP 60

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-GFP strain at 60 mins hyperosomotic shock


title: 016 W vs H - pABA

description: 016 W vs H - pABA


title: 011 W vs H - pABA

description: 011 W vs H - pABA

tag: Lactobacillus plantarum pNZ7021 (2) - pABA Cy3


title: Biological replicate 1 technical replicate 1 HOG1-GFP 0 mins

source_name: Cy5 labelled reference pool

tag: Pooled aRNA from all 6 samples in Bio replicate 1 technical replicate 1

source_name: B1 T1 HOG1-GFP 0 min

tag: Cy3 labelled aRNA created from total RNA isolated from the HOG1-GFP strain at 0 mins hyperosomotic shock


title: 008 W vs H + pABA

description: 008 W vs H + pABA

tag: Lactobacillus plantarum pNZ7026 (2) + pABA Cy3

tag: Lactobacillus plantarum pNZ7026 (3) + pABA Cy3


title: 002 W vs H + pABA

description: 002 W vs H + pABA

tag: Lactobacillus plantarum pNZ7026 (1) + pABA Cy3

tag: Lactobacillus plantarum pNZ7021 (2) + pABA Cy5


title: 018 W vs H +- pABA

description: 018 W vs H +- pABA


title: 020 W vs H +- pABA

description: 020 W vs H +- pABA

tag: Lactobacillus plantarum pNZ7026 ((1) - pABA Cy3

tag: Lactobacillus plantarum pNZ7021 (3) + pABA Cy5


title: 009 W vs H - pABA

description: 009 W vs H - pABA

tag: Lactobacillus plantarum pNZ7021 (1) - pABA Cy3

tag: Lactobacillus plantarum pNZ7026 (1) - pABA Cy5


title: 005 Wt vs Hyp + pABA

description: 005 Wt vs Hyp + pABA

tag: Lactobacillus plantarum pNZ7021 (3) + pABA Cy3

tag: Lactobacillus plantarum pNZ7026 (2) + pABA Cy5


title: 004 Wt vs Hyp + pABA

description: 004 Wt vs Hyp + pABA


title: 010 W vs H - pABA

description: 010 W vs H - pABA

tag: Lactobacillus plantarum pNZ7026 (1) - pABA Cy3

tag: Lactobacillus plantarum pNZ7021 (2) - pABA Cy5


title: 015 W vs H - pABA

description: 015 W vs H - pABA

tag: Lactobacillus plantarum pNZ7021 (1) - pABA Cy5


title: 006 Wt vs Hyp + pABA

description: 006 Wt vs Hyp + pABA

tag: Lactobacillus plantarum pNZ7021 (1) + pABA Cy5


title: 007 W vs H + pABA

description: 007 W vs H + pABA

tag: Lactobacillus plantarum pNZ7021 (2) + pABA Cy3


title: PDCUMNT vs ZFCUMNT_ref3

status: Public on May 30 2008

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

country: Germany

institute: Helmholtz Zentrum München GmbH

description: RNA isolation, Labelling, Hybridisation and Scanning procedure are described in the fields above

hyb_protocol: This hybridization mixture was placed on a prehybridised microarray, under a cover slip, placed into a hybridization chamber (Genetix) and immersed in a thermostatic bath at 42°C for at least 16 hours. After hybridization slides were washed in 40ml of 3x , 1x, 0.25x and 0.1x SSC at room temperature. For drying slides were placed in an empty 50 ml Falcon tube (Becton Dickinson, USA) and centrifuged at 4000m/s2. Beckers, J., Herrmann, F., Rieger, S., Drobyshev, A., Horsch, M., Hrabé de Angelis, M. and Seliger, B. (2005): Identification and validation of novel ERBB2 (Her2, NEU) targets including genes involved in angiogenesis. Int. J. Cancer 114: 590-597. Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas E, Schädler S, Erfle V, Beckers J, Schätzl H and Leib-Mösch C (2005): Cell Line Dependent RNA Expression Profiles of Prion-infected Mouse Neuronal Cells. JMB 349: 487-500 Seltmann M, Horsch M, Drobyshev A, Chen Y, Hrabé de Angelis M, Beckers J (2005): Assessment of a Systematic Expression Profiling. Approach in ENU-Induced Mouse Mutant Lines. Mam Genome 16 (1): 1-10

scan_protocol: Dried slides were scanned with a GenePix 4000A microarray scanner and the images were analyzed using the GenePix Pro3.0 image processing software (Axon Instruments, USA) Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas E, Schädler S, Erfle V, Beckers J, Schätzl H and Leib-Mösch C (2005): Cell Line Dependent RNA Expression Profiles of Prion-infected Mouse Neuronal Cells. JMB 349: 487-500 Seltmann M, Horsch M, Drobyshev A, Chen Y, Hrabé de Angelis M, Beckers J (2005): Assessment of a Systematic Expression Profiling. Approach in ENU-Induced Mouse Mutant Lines. Mam Genome 16 (1): 1-10

data_processing: Normalisation with GenePix Pro 6.0: Ratio based normalized data in each image so that the mean of ratio of means of all of the features is equal to 1. The VALUE column contains the log2 ratio of the result file after normalisation

organism: Mus musculus

source_name: PDCUMNT

label_protocol:
For labeling 20 µg of total RNA were used for reverse transcription and indirectly labeled with Cy3 or Cy5 fluorescent dye according the TIGR protocol (Hedge et al., 2000). Labeled cDNA was dissolved in 30 µl hybridization buffer (6x SSC, 0.5% SDS, 5x Denhardt’s solution and 50% formamide) and mixed with 30 µl of reference cDNA solution (pool from six control animals) labeled with the second dye.
HegdeP, Qi R, Abernathy R, Gay C, Dharap S, et al (2000): A concise guide to cDNA microarray analysis-II. Biotechniques 29: 548-562

extract_protocol: Total RNA was isolated just before processing for expression profiling. For preparation of total RNA individual organs were thawed in buffer containing chaotropic salt (RLT buffer, Qiagen) and homogenised using a Polytron homogeniser. Total RNA from individual samples was obtained according to manufacturer’s protocols using RNeasy Midi kits (Qiagen). The concentration was calculated from OD260/280 measurement and 2 µg RNA aliquots were run on a formaldehyde agarose gel to check for RNA integrity. The RNA was stored at -80°C in RNase free water (Qiagen).

tag: PDCUMNT Piezzo cloned B6D2F1 cumulus cell cloned embryo derived ES cell line

source_name: ZFCUMNT

tag: ZFCUMNT Zona-free cloned B6D2F1 cumulus cell cloned embryo derived ES cell line


title: 001 W vs H + pABA

description: 001 W vs H + pABA

tag: Lactobacillus plantarum pNZ7021 (1) + pABA Cy3

tag: Lactobacillus plantarum pNZ7026 (1) + pABA Cy5


title: 021 W vs H +- pABA

description: 021 W vs H +- pABA

tag: Lactobacillus plantarum pNZ7026 (2) + pABA Cy3


title: HM-1 NT vs ZFCUMNT_ref4

source_name: HM-1 NT

tag: HM-1 NT HM-1 NT-embryo derived ES cell line; HPRT-deficient; 129/SvJ


title: 012 W vs H - pABA

description: 012 W vs H - pABA

tag: Lactobacillus plantarum pNZ7026 (3) - pABA Cy3

tag: Lactobacillus plantarum pNZ7021 (3) - pABA Cy5


title: 003 Wt vs Hyp + pABA

description: 003 Wt vs Hyp + pABA

tag: Lactobacillus plantarum pNZ7026 (3) + pABA Cy5


title: HM-1 NT vs ZFCUMNT_ref3


title: PDCUMNT vs ZFCUMNT_ref1


title: PDCUMNT vs ZFCUMNT_ref2


title: PDCUMNT vs ZFCUMNT_ref4


title: #4 (B6) vs ZFCUMNT_ref2

tag: #4 (B6) B6D2F1 ES cell line


title: #4 (B6) vs ZFCUMNT_ref4


title: #4 (B6) vs B6PGA_ref2

tag: B6PGA B6D2F1 PGA ES cell line, parthenogenetic embryo derived ES cell line


title: #4 (B6) vs B6PGA_ref4


title: #4 (B6) vs ZFCUMNT_ref1


title: HM-1 NT vs ZFCUMNT_ref2


title: #4 (B6) vs B6NT_ref1

tag: B6NT B6D2F1 MEF NT-embryo derived ES cell line; B6D2F1


title: #4 (B6) vs ZFCUMNT_ref3


title: B6NT vs ZFCUMNT_ref4


title: #4 (B6) vs B6PGA_ref3


title: B6NT vs ZFCUMNT_ref2


title: #4 (B6) vs B6NT_ref2


title: #4 (B6) vs B6NT_ref3


title: B6NT vs ZFCUMNT_ref3


title: #4 (B6) vs B6NT_ref4


title: HM-1 NT vs ZFCUMNT_ref1


title: B6NT vs HM-1 NT_ref3


title: B6NT vs HM-1 NT_ref2


title: #4 (B6) vs B6PGA_ref1


title: B6NT vs ZFCUMNT_ref1


title: HM-1 vs ZFCUMNT_ref1

tag: HM-1 ES cell line; HPRT-deficient; 129/SvJ


title: HM-1 vs PDCUMNT_ref4


title: HM-1 vs ZFCUMNT_ref3


title: HM-1 vs ZFCUMNT_ref4


title: HM-1 vs PDCUMNT_ref2


title: HM-1 vs ZFCUMNT_ref2


title: HM-1 vs PDCUMNT_ref3


title: HM-1 vs PDCUMNT_ref1


title: B6NT vs HM-1 NT_ref4


title: B6NT vs HM-1 NT_ref1


title: HM-1 vs #4 (B6) _ref4


title: HM-1 vs B6 PGA_ref1


title: HM-1 vs B6 PGA_ref3


title: HM-1 vs B6NT_ref4


title: HM-1 vs B6NT_ref1


title: HM-1 vs B6 PGA_ref2


title: HM-1 vs #4 (B6) _ref3


title: HM-1 vs B6NT_ref2


title: HM-1 vs #4 (B6) _ref2


title: HM-1 vs #4 (B6) _ref1

tag: #4 (B6)	B6D2F1 ES cell line


title: HM-1 vs B6NT_ref3


title: HM-1 vs HM-1 NT_ref4


title: HM-1 vs B6 PGA_ref4


title: HM-1 vs HM-1 NT_ref1

tag: HM-1 NT	HM-1 NT-embryo derived ES cell line; HPRT-deficient; 129/SvJ


title: HM-1 vs HM-1 NT_ref3


title: HM-1 vs HM-1 NT_ref2


title: B205_orofarynx_1

status: Public on May 28 2008

city: Leiden

first: Jan

last: Oosting

address: Albinusdreef 2

department: Pathology

institute: Leiden University Medical Center

description: Gene expression data from squamous cell tumor

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on Affymetrix focus arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.

data_processing: The data files of all arrays were normalized using sequence information in Robust Multi_Chip Average (RMA) method.

label: biotin

organism: Homo sapiens

source_name: orofarynx, 1

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 10 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

tag: Location

value: orofarynx

tag: Metastasis


title: C206_larynx_1

source_name: larynx, 1

value: larynx


title: Expression profiling of U937 cells

city: Berlin

first: Pavlos

last: Chaitidis

address: Monbijoustr. 2

department: University Clinics Charite, Humboldt University

institute: Institute of Biochemistry

description:
Th2 Response of Human Peripheral Monocytes Involves Isoform-Specific Induction of Monoamine Oxidase-A
Pavlos Chaitidis, Ellen E. Billett, Valerie B. O’Donnell, Alexandra Bermudez Fajardo, Julia Fitzgerald, Ralf J. Kuban, Ute Ungethuem, and Hartmut Kuhn; The Journal of Immunology, 2004, 173: 4821–4827.
Availability in cell line catalogues: ATCC CRL 1593; IZSBS BS TCL73; DSMZ ACC 5; ICLC HTL94002;

hyb_protocol: According to Affymetrix

scan_protocol: According to Affymetrix

data_processing: Affymetrix MAS 4.0

label: According to Affymetrix

source_name: U937 as wild-type

label_protocol: According to Affymetrix

growth_protocol:
Incubation: 	at 37 °C with 5% CO2
Doubling time: 	about 30-40 hours

extract_protocol: According to QIAGEN (RNeasy-Kit)

treatment_protocol:
Medium: 	90% RPMI 1640 + 10% FBS
Subculture: 	maintain at 0.1-1.0 x 106 cells/ml; split 1:2 to 1:5 every 2-3 days; seed out at ca. 1 x 106 cells/ml
Harvest: 	saturation density at about 2.0 x 106 cells/ml
Storage: 	frozen with 70% medium, 20% FBS, 10% DMSO at about 4 x106 cells/ampoule

tag: U937 (human, Caucasian, pleural effusion, lymphoma, histiocytic)


title: C203_larynx_1


title: C205_larynx_1


title: C204_larynx_1


title: C103_larynx_0

source_name: larynx, 0


title: A204_mond_1

source_name: mouth, 1

value: mouth


title: Expression profiling of U937 cells stably transfected with ALOX15

description:
Th2 Response of Human Peripheral Monocytes Involves Isoform-Specific Induction of Monoamine Oxidase-A
Pavlos Chaitidis, Ellen E. Billett, Valerie B. O’Donnell, Alexandra Bermudez Fajardo, Julia Fitzgerald, Ralf J. Kuban, Ute Ungethuem, and Hartmut Kuhn;
The Journal of Immunology, 2004, 173: 4821–4827.

source_name: Modified U937

growth_protocol:
Incubation: 	at 37 °C with 5% CO2
Doubling time: 	about 15-20 hours

treatment_protocol:
Cell-culture:
Medium: 	90% RPMI 1640 + 10% FBS
Subculture: 	maintain at 0.1-1.0 x 10E6 cells/ml; split 1:2 to 1:5 every 2-3 days; seed out at ca. 1 x 10E6 cells/ml
Harvest: 	saturation density at about 2.0 x 10E6 cells/ml
Storage: 	frozen with 70% medium, 20% FBS, 10% DMSO at about 4 x10E6 cells/ampoule
Cell transfection:
The eukaryotic expression vector pcDNA 3 (Invitrogen Life Technologies, Leek, The Netherlands) bearing the complete coding region of the rabbit reticulocyte type 15-LOX1 were transfected into U937 cells using FuGENE 6, according to the manufacturer’s instructions.
Briefly, 2.5 x 10E5 cells/ml were plated on 35-mm plates and transfected with 2 µg of plasmid using 6 µl of FuGENE 6 reagent. After 72h exposure, the precipitate was removed and the cells were cultured for 4 weeks in selection medium supplemented with 0.5 mg/ml geneticin

tag: U937 (human, Caucasian, pleural effusion, lymphoma, histiocytic) stably transfected with ALOX15


title: SG_w1118_14APF_rep2

status: Public on May 29 2008

city: Fayetteville

first: Michael

last: Lehmann

state: AR

address: SCEN 601

department: Biological Sciences

institute: University of Arkansas

description: Salivary glands from w1118 animals heat shocked at 9.5 hours after puparium formation and dissected at 14 hours after puparium formation.

hyb_protocol: Hybridization was performed following the instructions provided in the Affymetrix Manual (Section 2), using a Fluidics Station 450 and the Midi_euk2 v3 protocol.

scan_protocol: GeneChips were scanned using the Affymetrix Genechip 3000 Scanner 7G and Genechip Operation Software (GCOS) v. 1.4.

data_processing: The data were processed and normalized using dChip 2006. The median intensity of the baseline assay used for normalization was 150.

organism: Drosophila melanogaster

source_name: salivary gland, heat shocked, dissected 14 hours after puparium formation

label_protocol: Biotin-labeled cRNA was produced using the One-Cycle Eukaryotic Target Labeling Assay recommended by Affymetrix. Labeling was carried out exactly as specified in the instructions manual, section 2 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).

growth_protocol: Animals were kept at 25 °C, except during heat shock period.

extract_protocol: Salivary glands were dissected on ice and directly transferred to TRIzol Reagent (Invitrogen, Carlsbad, California). To support lysis of the tissue, the mixture was subjected to vigorous vortexing. Total RNA was isolated according to the instructions of the manufacturer. As a last step of the extraction procedure, the RNA was purified using an RNeasy Minikit (Quiagen, Hilden, Germany).

treatment_protocol: Animals were subjected to a 30-min heat shock treatment at 37 °C at 9.5 hours after puparium formation. Salivary glands were dissected for RNA extraction at 14 hours after puparium formation.

tag: Salivary glands of strain w1118, dissected at 14 hours after puparium formation.


title: B203_orofarynx_1


title: B202_orofarynx_1


title: B102_orofarynx_0

source_name: orofarynx, 0


title: B101_orofarynx_0


title: Expression profiling of U937 cells stably transfected with control vector

source_name: Modified U937 cells

treatment_protocol:
Cell-culture:
Medium: 	90% RPMI 1640 + 10% FBS
Subculture: 	maintain at 0.1-1.0 x 10E6 cells/ml; split 1:2 to 1:5 every 2-3 days; seed out at ca. 1 x 10E6 cells/ml
Harvest: 	saturation density at about 2.0 x 10E6 cells/ml
Storage: 	frozen with 70% medium, 20% FBS, 10% DMSO at about 4 x10E6 cells/ampoule
Cell transfection:
The eukaryotic expression vector pcDNA 3 (Invitrogen Life Technologies, Leek, The Netherlands) bearing the empty vector (mock) were transfected into U937 cells using FuGENE 6, according to the manufacturer’s instructions.
Briefly, 2.5 x 10E5 cells/ml were plated on 35-mm plates and transfected with 2 µg of plasmid using 6 µl of FuGENE 6 reagent. After 72h exposure, the precipitate was removed and the cells were cultured for 4 weeks in selection medium supplemented with 0.5 mg/ml geneticin

tag: U937 (human, Caucasian, pleural effusion, lymphoma, histiocytic) stable transfected with control vector (pcDNA 3, Invitrogen Life Technologies, Leek, The Netherlands)


title: A202_mond_1


title: C105_larynx_0


title: SG_1P{hsGAL4}89(S)_14APF_rep1

description: Salivary glands from animals carrying one copy of the heat-inducible transgene P{GAL4-Hsp70.PB}89-2-1, heat shocked at 9 hours after puparium formation, and dissected at 14 hours after puparium formation.

source_name: GAL4-expressing salivary glands, heat shocked, dissected 14 hours APF

treatment_protocol: Animals were subjected to a 30-min heat shock treatment at 37 °C at 9 hours after puparium formation. Salivary glands were dissected for RNA extraction at 14 hours after puparium formation.

tag: Salivary glands of animals carrying one copy of the heat-inducible transgene P{GAL4-Hsp70.PB}89-2-1, heat shocked, dissected at 14 hours after puparium formation.


title: B106_orofarynx_0


title: B105_orofarynx_0


title: A201_mond_1


title: A206_mond_1


title: C101_larynx_0


title: SG_w1118_14APF_rep1


title: A105_mond_0

source_name: mouth, 0


title: A107_mond_0


title: A104_mond_0


title: C104_larynx_0


title: Fusion:Yes, Deletion:No (prostate_3900495)

city: New York

first: Mark

middle: A.

last: Rubin

state: NY

address: 1300 York Avenue Room C 410-A

department: Pathology and Laboratory Medicine

institute: Weill Cornell Medical Center

description: no additional

hyb_protocol: Refer to the Illumina DASL Assay System Manual. Hybridization was performed for 4 DASL assay oligo pool (DAP)s, DAP1~4, including 1536 probes each on 4 separate microarrays.

data_processing: For each probe, Red and Green intensities are computed as average of single bead values. The final signal is the sum of the Red and Green intensities. Data were median-centered by subtraction of median (log2 scale) within sample and within DAP, and median set to a target value computed as the overall within DAP median. Data were then normalized using the qspline algorithm (on the log2 intensity scale) with reference distribution computed as the marginal (always within DAP) mean of every gene.

label: Cy3 and Cy5

source_name: primary prostate cancer

extract_protocol: Using 3 biopsy cores (0.6µm each) taken from formalin-fixed paraffin-embeded tissue blocks, total RNA was extracted using Trizol LS reagent (Invitrogen).


title: Fusion:No, Deletion:No (prostate_R1981852)

description: no additional

scan_protocol: Refer to the Illumina DASL Assay System Manual


title: Fusion:Yes, Deletion:No (prostate_C3533743)

description: no additional

label_protocol: Refer to the Illumina DASL Assay System Manual


title: Fusion:No, Deletion:No (prostate_C2595457)

description: no additional


title: Fusion:No, Deletion:No (prostate_4217152)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_R2709313)

description: no additional

tag: FUSION:

tag: DELETION:


title: Fusion:No, Deletion:No (prostate_R3623364)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_C1139447)

description: no additional


title: Fusion:No, Deletion:No (prostate_3600229)

description: no additional


title: Fusion:No, Deletion:No (prostate_C1786815)

description: no additional


title: Fusion:No, Deletion:No (prostate_3552676)

description: no additional


title: Fusion:No, Deletion:No (prostate_C1266564)

description: no additional


title: Fusion:No, Deletion:No (prostate_R3725708)

description: no additional


title: Fusion:No, Deletion:No (prostate_C3158474)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_3529142)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_C2663151)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_C3351732)

description: no additional


title: Fusion:Yes, Deletion: unknown (prostate_3489560)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_3414033)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_3478398)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_3472011)

description: no additional


title: Fusion:No, Deletion:No (prostate_3442646)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_3603389)

description: no additional


title: Fusion:No, Deletion:No (prostate_3287990)

description: no additional


title: Fusion:No, Deletion:No (prostate_C1317904)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_3293315)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_3285506)

description: no additional


title: Fusion:No, Deletion:No (prostate_3178403)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2851866)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_3036077)

description: no additional


title: Fusion:No, Deletion:No (prostate_3064129)

description: no additional


title: Fusion:No, Deletion:No (prostate_3500855)

description: no additional


title: Fusion:No, Deletion:No (prostate_3063238)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_3099769)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_2859706)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2870301)

description: no additional


title: Fusion:No, Deletion:No (prostate_3148139)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_2794844)

description: no additional


title: Fusion:No, Deletion:No (prostate_2834458)

description: no additional


title: Fusion:Yes, Deletion: unknown (prostate_2964505)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2621700)

description: no additional


title: Fusion:No, Deletion:No (prostate_2645342)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_2522181)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2428026)

description: no additional


title: Fusion:No, Deletion:No (prostate_2706827)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2411454)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2428788)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_3107992)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_2469615)

description: no additional


title: Fusion:No, Deletion:No (prostate_2390773)

description: no additional


title: Fusion:No, Deletion:No (prostate_2473980)

description: no additional


title: Fusion:No, Deletion:No (prostate_2506210)

description: no additional


title: Fusion:Yes, Deletion: unknown (prostate_2927390)

description: no additional


title: Fusion:No, Deletion:No (prostate_2355997)

description: no additional


title: Fusion:Yes, Deletion: unknown (prostate_2437135)

description: no additional


title: Fusion:No, Deletion:No (prostate_2344270)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_2516355)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_2272451)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2339744)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2332668)

description: no additional


title: Fusion:No, Deletion:No (prostate_2204000)

description: no additional


title: Fusion:No, Deletion:No (prostate_2191499)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_2196708)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_2174891)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_1981330)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_1901852)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2106472)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_2111629)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2114475)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_1888698)

description: no additional


title: Fusion:No, Deletion:No (prostate_2089436)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_2104676)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_1819204)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_2013096)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_2042208)

description: no additional


title: Fusion:No, Deletion:No (prostate_2305348)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_1860321)

description: no additional


title: Fusion:No, Deletion:No (prostate_1867694)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_1797054)

description: no additional


title: Fusion:No, Deletion:No (prostate_1812311)

description: no additional


title: Fusion:No, Deletion:No (prostate_2126440)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_1724455)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_1789943)

description: no additional


title: Fusion:Yes, Deletion: unknown (prostate_2157652)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_1703980)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_1714448)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_1681181)

description: no additional


title: Fusion:No, Deletion:No (prostate_1841131)

description: no additional


title: Fusion:No, Deletion:No (prostate_1670815)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_1616422)

description: no additional


title: Fusion:No, Deletion:No (prostate_1675210)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_1672851)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_1615161)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_1403552)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_1369034)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_1423640)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_1258113)

description: no additional


title: Fusion:No, Deletion:No (prostate_1366001)

description: no additional


title: Fusion:No, Deletion:No (prostate_1265345)

description: no additional


title: Fusion:No, Deletion:No (prostate_1257303)

description: no additional


title: Fusion:No, Deletion: unknown (prostate_1345551)

description: no additional


title: Fusion:No, Deletion:No (prostate_1134167)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_1306957)

description: no additional


title: Fusion:No, Deletion:No (prostate_1342776)

description: no additional


title: Fusion:No, Deletion:No (prostate_1252834)

description: no additional


title: Fusion:Yes, Deletion: unknown (prostate_1132924)

description: no additional


title: Fusion:No, Deletion:No (prostate_1420826)

description: no additional


title: Fusion: Yes, Deletion: No (prostate_44396)

description: no additional

tag: FUSION

value: YES

tag: DELETION

value: NO


title: Fusion: No, Deletion: unknown (prostate_44164)

description: no additional


title: Fusion: No, Deletion: No (prostate_44222)

description: no additional


title: Fusion: Yes, Deletion: Yes (prostate_44040)

description: no additional


title: Fusion: No, Deletion: No (prostate_44032)

description: no additional


title: Fusion: Yes, Deletion: unknown (prostate_44248)

description: no additional


title: Fusion: No, Deletion: No (prostate_44255)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_44016)

description: no additional


title: Fusion:Yes, Deletion:No (prostate_1208032)

description: no additional


title: Fusion: Yes, Deletion: unknown (prostate_43984)

description: no additional


title: Fusion: No, Deletion: No (prostate_43976)

description: no additional


title: Fusion: Yes, Deletion: No (prostate_43737)

description: no additional


title: Fusion: Yes, Deletion: Yes (prostate_43851)

description: no additional


title: Fusion: Yes, Deletion: unknown (prostate_43372)

description: no additional


title: Fusion: Yes, Deletion: No (prostate_43307)

description: no additional


title: Fusion: Yes, Deletion: No (prostate_43497)

description: no additional


title: Fusion: No, Deletion: No (prostate_43158)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_43133)

description: no additional


title: Fusion: No, Deletion: No (prostate_43489)

description: no additional


title: Fusion: No, Deletion: No (prostate_42986)

description: no additional


title: Fusion: No, Deletion: No (prostate_43539)

description: no additional


title: Fusion: No, Deletion: No (prostate_41483)

description: no additional


title: Fusion: No, Deletion: No (prostate_42804)

description: no additional


title: Fusion: Yes, Deletion: Yes (prostate_41442)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_41764)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_43810)

description: no additional


title: Fusion: No, Deletion: No (prostate_42739)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_40287)

description: no additional


title: Fusion: No, Deletion: No (prostate_41939)

description: no additional


title: Fusion: Yes, Deletion: unknown (prostate_40030)

description: no additional


title: Fusion: Yes, Deletion: No (prostate_42994)

description: no additional


title: Fusion: No, Deletion: No (prostate_40873)

description: no additional


title: Fusion: No, Deletion: No (prostate_40691)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_33154)

description: no additional


title: Fusion: No, Deletion: No (prostate_41137)

description: no additional


title: Fusion: Yes, Deletion: Yes (prostate_41319)

description: no additional


title: Fusion: No, Deletion: No (prostate_41707)

description: no additional


title: Fusion:Yes, Deletion:Yes (prostate_3177375)

description: no additional


title: Fusion: Yes, Deletion: Yes (prostate_31104)

description: no additional


title: Fusion: No, Deletion: No (prostate_32565)

description: no additional


title: Fusion: No, Deletion: No (prostate_21881)

description: no additional


title: Fusion: No, Deletion: No (prostate_32839)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_33522)

description: no additional


title: Fusion: No, Deletion: No (prostate_21782)

description: no additional


title: Fusion: No, Deletion: No (prostate_30262)

description: no additional


title: Fusion: Yes, Deletion: Yes (prostate_21170)

description: no additional


title: Fusion: No, Deletion: No (prostate_20479)

description: no additional


title: Fusion: No, Deletion: No (prostate_13602)

description: no additional


title: Fusion: No, Deletion: No (prostate_11866)

description: no additional


title: Fusion: No, Deletion: No (prostate_12435)

description: no additional


title: Fusion: No, Deletion: No (prostate_12591)

description: no additional


title: Fusion: No, Deletion: No (prostate_12989)

description: no additional


title: Fusion: Yes, Deletion: unknown (prostate_30221)

description: no additional


title: Fusion: No, Deletion: No (prostate_10942)

description: no additional


title: Fusion: No, Deletion: No (prostate_13458)

description: no additional


title: Fusion: Yes, Deletion: Yes (prostate_13425)

description: no additional


title: Fusion: No, Deletion: No (prostate_10330)

description: no additional


title: Fusion: No, Deletion: No (prostate_10538)

description: no additional


title: Fusion: No, Deletion: No (prostate_10405)

description: no additional


title: Fusion: No, Deletion: No (prostate_10439)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_20594)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_1966)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_844)

description: no additional


title: Fusion: No, Deletion: No (prostate_10785)

description: no additional


title: Fusion: Yes, Deletion: unknown (prostate_1081)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_1545)

description: no additional


title: Fusion: unknown, Deletion: unknown (prostate_786)

description: no additional


title: Fusion: No, Deletion: unknown (prostate_794)

description: no additional


title: Fusion: Yes, Deletion: No (prostate_42481)

description: no additional information


title: Fusion: No, Deletion: No (prostate_42630)

description: no additional information


title: Fusion: No, Deletion: No (prostate_42762)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_59)

description: no additional


title: Fusion: No, Deletion: No (prostate_42093)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_42036)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_41996)

description: no additional information


title: Fusion: No, Deletion: No (prostate_41871)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_41566)

description: no additional information


title: Fusion: No, Deletion: No (prostate_41665)

description: no additional information


title: Fusion: No, Deletion: No (prostate_41434)

description: no additional information


title: Fusion: No, Deletion: No (prostate_41590)

description: no additional information


title: Fusion: Yes, Deletion: Yes (prostate_41533)

description: no additional information


title: Fusion: No, Deletion: No (prostate_42135)

description: no additional information


title: Fusion: Yes, Deletion: Yes (prostate_40931)

description: no additional information


title: Fusion: No, Deletion: No (prostate_41368)

description: no additional information


title: Fusion: No, Deletion: No (prostate_41004)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_41335)

description: no additional information


title: Fusion: Yes, Deletion: unknown (prostate_40196)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_40261)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_40741)

description: no additional information


title: Fusion: No, Deletion: No (prostate_40477)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_33423)

description: no additional information


title: Fusion: No, Deletion: No (prostate_33084)

description: no additional information


title: Fusion: Yes, Deletion: Yes (prostate_33431)

description: no additional information


title: Fusion: No, Deletion: No (prostate_33373)

description: no additional information


title: Fusion: No, Deletion: No (prostate_33290)

description: no additional information


title: Fusion: No, Deletion: No (prostate_33506)

description: no additional information


title: Fusion: No, Deletion: No (prostate_41376)

description: no additional information


title: Fusion: No, Deletion: No (prostate_32714)

description: no additional information


title: Fusion: No, Deletion: No (prostate_32805)

description: no additional information


title: Fusion: No, Deletion: No (prostate_32540)

description: no additional information


title: Fusion: No, Deletion: No (prostate_32201)

description: no additional information


title: Fusion: Yes, Deletion: Yes (prostate_32219)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_40170)

description: no additional information


title: Fusion: No, Deletion: No (prostate_31856)

description: no additional information


title: Fusion: No, Deletion: No (prostate_31989)

description: no additional information


title: Fusion: Yes, Deletion: Yes (prostate_31617)

description: no additional information


title: Fusion: No, Deletion: No (prostate_31351)

description: no additional information


title: Fusion: Yes, Deletion: No (prostate_31559)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_30627)

description: no additional information


title: Fusion: No, Deletion: No (prostate_32912)

description: no additional information


title: Fusion: No, Deletion: No (prostate_32375)

description: no additional information


title: Fusion: Yes, Deletion: No (prostate_31914)

description: no additional information


title: Fusion: No, Deletion: No (prostate_21626)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_30445)

description: no additional information


title: Fusion: No, Deletion: No (prostate_21634)

description: no additional information


title: Fusion: No, Deletion: No (prostate_21667)

description: no additional information


title: Fusion: No, Deletion: No (prostate_21758)

description: no additional information


title: Fusion: No, Deletion: No (prostate_21014)

description: no additional information


title: Fusion: No, Deletion: No (prostate_20982)

description: no additional information


title: Fusion: No, Deletion: No (prostate_30320)

description: no additional information


title: Fusion: No, Deletion: No (prostate_21568)

description: no additional information


title: Fusion: No, Deletion: No (prostate_21857)

description: no additional information


title: Fusion: No, Deletion: No (prostate_20701)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_20503)

description: no additional information


title: Fusion: No, Deletion: No (prostate_20875)

description: no additional information


title: Fusion: No, Deletion: No (prostate_21477)

description: no additional information


title: Fusion: Yes, Deletion: Yes (prostate_20420)

description: no additional information


title: Fusion: Yes, Deletion: No (prostate_21055)

description: no additional information


title: Fusion: No, Deletion: No (prostate_20685)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_21097)

description: no additional information


title: Fusion: unknown, Deletion: unknown (prostate_12641)

description: no additional information


title: Fusion: No, Deletion: No (prostate_13003)

description: no additional information


title: Fusion: Yes, Deletion: No (prostate_20073)

description: no additional information


title: Fusion: No, Deletion: No (prostate_13037)

description: no additional information


title: Fusion: No, Deletion: No (prostate_13276)

description: no additional information


title: Fusion: Yes, Deletion: Yes (prostate_12633)

description: no additional information


title: Fusion: No, Deletion: No (prostate_12997)

description: no additional information


title: Fusion: No, Deletion: No (prostate_20388)

description: no additional information


title: Fusion: No, Deletion: No (prostate_13516)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_12492)

description: no additional information


title: Fusion: No, Deletion: No (prostate_12518)

description: no additional information


title: Fusion: No, Deletion: No (prostate_12484)

description: no additional information


title: Fusion: No, Deletion: No (prostate_20321)

description: no additional information


title: Fusion: No, Deletion: No (prostate_12344)

description: no additional information


title: Fusion: No, Deletion: No (prostate_11825)

description: no additional information


title: Fusion: No, Deletion: No (prostate_13102)

description: no additional information


title: Fusion: No, Deletion: No (prostate_11106)

description: no additional information


title: Fusion: No, Deletion: No (prostate_11619)

description: no additional information


title: Fusion: No, Deletion: No (prostate_12062)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_10215)

description: no additional information


title: Fusion: No, Deletion: No (prostate_11759)

description: no additional information


title: Fusion: No, Deletion: No (prostate_32896)

description: no additional


title: Fusion: No, Deletion: No (prostate_10983)

description: no additional information


title: Fusion: No, Deletion: No (prostate_11908)

description: no additional information


title: Fusion: No, Deletion: No (prostate_1727)

description: no additional information


title: Fusion: No, Deletion: No (prostate_10165)

description: no additional information


title: Fusion: No, Deletion: No (prostate_10421)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_2469)

description: no additional information


title: Fusion: No, Deletion: No (prostate_2444)

description: no additional information


title: Fusion: No, Deletion: No (prostate_2154)

description: no additional information


title: Fusion: No, Deletion: No (prostate_11080)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_2048)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_1446)

description: no additional information


title: Fusion: unknown, Deletion: unknown (prostate_1404)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_208)

description: no additional information


title: Fusion: unknown, Deletion: unknown (prostate_1065)

description: no additional information


title: CHCC2862_Aerobic/Respiratory_Repl

status: Public on May 27 2008

city: Cadorago

middle: Bastian

last: Pedersen

state: CO

address: Via Manzoni 29

department: R&D

country: Italy

institute: Sacco Srl

description: Comparison of the physiological response of aerobic to respiratory growth with Lactococcus lactis subsp. lactis CHCC2862. NOTE: Compared to GSM202337 this array is dye-swapped.

hyb_protocol: According to the Amersham protocol the Cy3 and Cy5 labelled cDNA were combined and lyophilized. Hereafter they were resuspended in 7uL of nuclease free water and, 5uL of hybdridization buffer (4x), and 8uL of formamide was added. This 20uL solution was applied to the array under a 24x24mm LifterSlip (Erie Scientific Company, Portsmouth, NH) and put in an airtight box containing paper towels soaked with NaCl-saturated water. Arrays were hybridized for 16-20h at 42ºC, and then washed using the UltraGAPS protocol (Corning B.V.).

scan_protocol: Arrays were scanned using the GenePix 4100A Personal Scanner with GenePix Pro 6.0 software (Axon Instruments Inc., Union City, CA). The photo multiplier tube (PMT) sensitivity values of the scan were adjusted around 660 for Cy5 and 520 for Cy3 to obtain only few saturated spots on each array and about equal total signal for each dye.

data_processing:
A grid with 110um spots was laid over the array and was manually adjusted to fit small irregularities in spotting. Spots clearly covered by for example dust particles were flagged “Bad”. The intensity of all spots was then calculated using the “Analyze” function. Spots with very weak signals in both channels were also flagged “Bad”. These were spots where the signal-to-noise ratio [SNR] < 1 in both channels, or where [% > B + 2SD] < 20 in both channels. All other spots were considered “Found” and had a “Flag” value of >= 0. All data was exported from GenePix Pro in tab-delimited format.
In the further analysis the variables of feature (spot) intensity minus the background intensity around the spot was used, i.e. [F635 median – B635]=Cy5 and [F532 median – B532]=Cy3. The log2(ratio) of this was calculated as log2(Cy5/Cy3). Some spots may have a significant signal in one channel but no signal in the other channel. The latter may result in a negative value due to noise and thus and illegal log2(ratio). To avoid this a macro in Excel 2000 (Microsoft Corporation, Redmond, WA) was used to change [F635 median – B635] and [F532 median – B532] values of <10 to 10 for “Found” spots (the signal scale goes from 0, no signal, to 65535, saturated signal). For these spots the log2(ratio) was re-calculated. The corrected data was exported from Excel and imported into Acuity 4.0 (Axon) where they were ratio-normalized based on the “Found” spots.
In Acuity a dataset was created. For each array only genes were included where 1) at least 3 of the 4 replicate spots were “Found”, 2) the standard deviation was <0.8 among “Found” spots, and 3) <30% of the pixels in “Found” spots, in both channels, were saturated.

organism: Lactococcus lactis subsp. lactis

source_name: Respiratory

label_protocol: 10ug of total RNA was lyophilized in a speed-vac. The copying into cDNA and labeling with either Cy3 (reference) or Cy5 (test) was done using the CyScribe Post-Labelling Kit using manufacturer’s protocol (Amersham Biosciences, Hillerød, Denmark). 1uL of random nonamer was used for priming.

extract_protocol: At OD 1.0 samples of 0.67mL were collected in 1.33mL RNAprotect according to protocol (QIAGEN, Valencia, CA). Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) except for the following: the 200uL TE buffer contained 15mg/mL of lysozyme, 15uL of proteinase K at 20mg/mL, and 2uL of mutanolysin at 25U/uL with vortexing every 10s at 37ºC for the 2min. The quality and concentration of RNA was determined using the RNA 6000 Nano Kit on the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Routinely, 20-50ug of total RNA was obtained. Only high quality total RNA, as indicated by two sharp rRNA peaks, was used.

tag: Respiratory

value: M17 + 0.5% lactose + 5mg/L hemin, agitation at 170rpm. Harvest at OD600=1.0.

source_name: Aerobic

tag: Aerobic

value: M17 + 0.5% lactose, agitation at 170rpm. Harvest at OD600=1.0.


title: CHCC2862_Respiratory/Static_Repl

description: Comparison of the physiological response of respiratory to static growth with Lactococcus lactis subsp. lactis CHCC2862

source_name: Static

tag: Static

value: M17 + 0.5% lactose, no agitation. Harvest at OD 1.0.

value: M17 + 0.5% + 5mg/L hemin, agitation at 170rpm. Harvest at OD600 1.0.


title: CHCC2862_Aerobic/Static

description: Comparison of the physiological response of aerobic to static growth with Lactococcus lactis subsp. lactis CHCC2862

hyb_protocol: According to the Amersham protocol the Cy3 and Cy5 labelled cDNA were combined and lyophilized. Hereafter they were resuspended in 7uL of nuclease free water and, 5uL of hybdridization buffer (4x), and  8uL of formamide was added. This 20uL solution was applied to the array under a 24x24mm LifterSlip (Erie Scientific Company, Portsmouth, NH) and put in an airtight box containing paper towels soaked with NaCl-saturated water. Arrays were hybridized for 16-20h at 42ºC, and then washed using the UltraGAPS protocol (Corning B.V.).

value: M17 + 0.5% lactose, no agitation. Harvested at OD600=1.0.

value: M17 + 0.5% lactose, agitation at 170rpm. Harvested at OD600=1.0.


title: CHCC2862_Respiratory/Static


title: Fusion: Yes, Deletion: unknown (prostate_307)

description: no additional information


title: Fusion: No, Deletion: unknown (prostate_10082)

description: no additional information


title: CHCC2862_Aerobic/Static_Repl


title: CHCC2862_Respiratory/Aerobic

description: Comparison of the physiological response of respiratory to aerobic growth with Lactococcus lactis subsp. lactis CHCC2862


title: PCAR_acetoin_FeNTA_BR1A

status: Public on May 22 2008

city: Amherst

first: Derek

last: Lovley

state: MA

address: University of Massachusetts at Amherst, Dept. of Microbiology, Morrill IV north room 202

department: Microbiology

institute: University of Massachusetts Amherst

description: total RNA was amplified using the Ambion MessageAmp II- Bacteria kit for polyadenylation and subsequent amplification of total RNA. aRNA was ethanol precipitated and resuspended in nuclease-free water to a total concentration of ~1 ug/ul.

hyb_protocol: Combimatrix 100 ul cap protocol.

scan_protocol: Slide was scanned using GenePix software and a GenePix 4000b scanner.

data_processing:
The raw total intensities were treated as follows to generate the M Log2 ratios for this work. Background signal was calculated from a set of negative control spots incorporated into the array. As the negative control sequences are not design specific (ie. are found in all of the ESOAs independent of the organism being sampled) some will inevitably demonstrate strong cross hybridization with the sample of interest. To avoid this issue the negative control spots were ranked according to their total intensity. The average of the lowest 30% was calculated. Background signal was calculated as this mean + 2 standard deviations. The background signal was then subtracted from the total intensity for each spot. Probes whose signal was not greater than zero after background subtraction were omitted from further analysis. For the remaining data, the logged ratios [M=log2 (Ex/Ct)] are then calculated and Lowess global normalization is performed. During the data preprocessing, M vs. A plots before and after normalization, and side-by-side box plots for all arrays are used to assess array quality.(3) LIMMA mixed model analysis (R-package LIMMA(2))is applied to the normalized logged ratios to identify differentially expressed genes. Specifically, biological replicates are treated as a randomized block to allow for modeling correlations within them. LIMMA calculates a moderated t statistic for each probe, which is the ratio of the estimated mean logged intensity ratio and the Empirical Bayes estimate of its standard error. The P-value of each oligo is then corrected for multiple comparisons according to Benjamini and Hockberg's procedure(1) to control the false discovery rate. Oligos are ranked according to the adjusted P-values and were called differentially expressed if the adjusted P-values are < 0.001. Note that multiple oligonucleotide probes from the same gene are analyzed separately and a gene is called differentially expressed if at least half of its probes are called differentially expressed.
1. Benjamini, Y., and Y. Hochberg. 1995. Controlling the False Discovery Rate - a Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B-Methodological 57:289-300.
2. Smyth, G. K. 2004. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:Article3.
3. Smyth, G. K., and T. Speed. 2003. Normalization of cDNA microarray data. Methods 31:265-73.

organism: Syntrophotalea carbinolica

source_name: Pelobacter carbinolicus grown on EtOH/FeNTA

label_protocol: Micromax ASAP labeling kit on 10 ug aRNA.

extract_protocol: For reference see:In situ expression of nifD in Geobacteraceae in subsurface sediments. Appl Environ Microbiol. 2004 Dec;70(12):7251-9. PMID: 15574924

tag: Pelobacter carbinolicus was grown in batch culture containing ethanol as the electron donor and Fe-NTA as the electron acceptor.

source_name: Pelobacter carbinolicus grown on acetoin

label_protocol: Micromax ASAP RNA labeling kit on 10 ug aRNA.

tag: Pelobacter carbinolicus was grown in batch culture containing acetoin.


title: PCAR_acetoin_FeNTA_BR2A


title: PCAR_acetoin_FeNTA_BR2B


title: Whole Testis replica_5

status: Public on May 19 2008

city: Cambridge

first: Scott

last: Jelinsky

address: 610 Main Street

department: Inflammation and Immunology

institute: Pfizer

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Rat Genome Array RG230 2.0. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the GeneChip® Scanner 3000 with AutoLoader.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.

label: Biotin

organism: Rattus norvegicus

source_name: Rattus norvegicus Whole Testis

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Sprague Dawley rats 55-60 days of age were used for the collection of seminiferous tubules at defined stages of the cycle and for the isolation of Sertoli cells, pachytene spermatocytes and round spermatids.  Rats 18-20 days of age were used for the isolation of a pool of spermatogonia and early spermatocytes.

extract_protocol: Trizol extraction of total RNA follwed by further purification with RNAeasy was performed according to the manufacturer's instructions.

treatment_protocol: Seminiferous tubules at the following stages or groups of stages were isolated by transillumination-assisted microdissection: I, II-III, IV-V, VI, VIIa,b,  VIIc,d, VIII, IX-XI, XII, XIII-XIV. Twenty-five segments of tubules, 2 m in length, were collected at each stage or group of stages from each rat.  Tubules from two separate animals were pooled to form one sample from each stage or groups of stages.  A total of 5 independent samples per stage or groups of stages were analyzed. Mature Sertoli cells were isolated to about 85% purity (n=4). Three groups of germ cells were isolated by centrifugal elutriation from enzyme-dispersed testes: round spermatids (n=4), pachytene spermatocytes (n=4) and spermatogonia plus early spermatocytes (n=3) . Purity of these cells has been shown to vary between 85% to 95%

tag: Strain:Sprague Dawley

tag: Age:55-60 days


title: Whole Testis replica_3


title: Whole Testis replica_2


title: PCAR_acetoin_FeNTA_BR1B


title: Spermatogonia Plus Early Spermatocytes  Replica_2

source_name: Rattus norvegicus Spermatogonia and early spermatocytes

tag: Age:18-20 days


title: PCAR_acetoin_FeNTA_BR3B


title: Whole Testis replica_4


title: PCAR_acetoin_FeNTA_BR3A


title: mp53_polysomal_Rep8

status: Public on May 25 2008

type: other

city: Rochester

first: Hartmut

last: Land

address: 601 Elmwood Avenue, Box 633

department: Biomedical Genetics

institute: University of Rochester Medical Center

description: Refer to submitted paper, McMurray et al.

hyb_protocol: The cRNA yield was fragmented and hybridization cocktails were prepared using Affymetrix standard protocol for eukaryotic target hybridization.  Targets were hybridized to Affymetrix Mouse Genome 430 2.0 Expression Arrays at 45°C for 16 hours, washed and stained using Affymetrix Fluidics protocol EukGE-WS2v4_450 in the Fluidics Station 450.

scan_protocol: Arrays were scanned with the Affymetrix GeneChip Scanner 3000.

data_processing: Expression values from the 50 microarrays processed were obtained using the RMA procedure with background correction in Bioconductor (http://www.bioconductor.org).

molecule: other

source_name: mp53-expressing cells, polysomal RNA

label_protocol: Five micrograms of RNA was reverse transcribed and labeled using the mAMP kit (Ambion), with the 1x amplification protocol.

growth_protocol: Four polyclonal cell populations, control (Bleo/Neo), mp53 (p53175H/Neo), Ras (Bleo/RasV12) and mp53/Ras (p53175H/RasV12) were derived by retroviral infection of low-passage polyclonal young adult mouse colon (YAMC) cells.  YAMC cells (a gift from R. Whitehead and A.W. Burgess) derived from the Immorto-mouse (aka H-2Kb/tsA58 transgenic mouse) expressing temperaturesensitive simian virus 40 large T (tsA58) under the control of an interferon γ-inducible promoter were maintained at the permissive temperature (33°C) for large T in the presence of interferon γ to support conditional immortalization in vitro. This permits expansion of the cells in tissue culture. In contrast, exposure of YAMC cells to the non-permissive temperature for large T (39°C) in the absence of interferon γ leads to growth arrest followed by cell death, indicating the absence of spontaneous immortalizing mutations in the cell population. The cells were cultured on Collagen IV-coated dishes (1μg/cm2 for 1.5 hr at room temp; Sigma) in RPMI 1640 medium (Invitrogen) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone), 1×ITS-A (Invitrogen), 2.5 μg/ml gentamycin (Invitrogen), and 5 U/ml interferon γ (R&D Systems). All experiments testing the effects of RasV12 and p53175H were carried out at the non-permissive temperature for large T function (39°C) and in the absence of interferon γ.

extract_protocol: Polysomal RNA was harvested from YAMC, bleo/neo, mp53/neo, bleo/Ras and mp53/Ras cells to obtain gene expression profiles reflective of protein synthesis rates.  Cells were lysed in Extraction Buffer (50 mM MOPS, 15 mM MgCl, 150 mM NaCl, 0.5% Triton X-100 with 100 ug/mL cycloheximide, 1 mg/mL heparin, 200U RNAsin (2 uL/mL of buffer), 2mM PMSF).  Supernatants were applied to 10-50% sucrose gradients, centrifuged at 36,000 rpm for 2 hr at 4°C and fractions were collected using an ISCO gradient fractionator reading absorbance at 254 nm.  Polysome containing fractions were pooled and RNA was purified using the RNeasy Mini Kit (Qiagen) following the standard protocol for animal cells, except that sucrose fractions were mixed with 3.5 volumes Buffer RLT before binding to the RNeasy column.  RNA was on-column DNase digested as part of the RNeasy RNA extraction protocol.

treatment_protocol: RNA was harvested from ten replicates for each cell population grown in non-permissive conditions for 48 hr, followed by 24 hr in media with 0% FBS to maximize the contribution of oncogenic signaling to gene expression.  RNA was collected while cells were sub-confluent and all cell populations were actively cycling.

tag: Refer to Whitehead, R. H., VanEeden, P. E., Noble, M. D., Ataliotis, P. & Jat, P. S. Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. Proc Natl Acad Sci U S A 90, 587-91 (1993) and Xia, M. & Land, H. Tumor suppressor p53 restricts Ras stimulation of RhoA and cancer cell motility. Nat Struct Mol Biol 14, 215-23 (2007).


title: Spermatogonia Plus Early Spermatocytes  Replica_1


title: Pachytene Spermatocytes  Replica_3

source_name: Rattus norvegicus Pachytene Spermatocytes


title: Whole Testis replica_1


title: Pachytene Spermatocytes  Replica_1


title: Spermatogonia Plus Early Spermatocytes  Replica_3


title: Round Spermatid  Replica_2

source_name: Rattus norvegicus Round Spermatid


title: Round Spermatid  Replica_1


title: Pachytene Spermatocytes  Replica_4


title: Sertoli Cells  Replica_1

source_name: Rattus norvegicus Sertoli cells


title: Sertoli Cells  Replica_3


title: Round Spermatid  Replica_4


title: Pachytene Spermatocytes  Replica_2


title: Round Spermatid  Replica_3


title: Sertoli Cells  Replica_2


title: Seminiferous Tubules Stage (XIII-XIV) Replica_2

source_name: Rattus norvegicus Seminiferous Tubules at Stages XIII-XIV


title: Sertoli Cells  Replica_4


title: Seminiferous Tubules Stage (XIII-XIV) Replica_5


title: Seminiferous Tubules Stage (XIII-XIV) Replica_4


title: Seminiferous Tubules Stage (IX-XI) Replica_2

source_name: Rattus norvegicus Seminiferous Tubules at Stages IX-XII


title: Seminiferous Tubules Stage (XII) Replica_4

source_name: Rattus norvegicus Seminiferous Tubules at Stage XII


title: Seminiferous Tubules Stage (XIII-XIV) Replica_3


title: Seminiferous Tubules Stage (XII) Replica_3


title: Seminiferous Tubules Stage (XII) Replica_1


title: Seminiferous Tubules Stage (VIII) Replica_4

source_name: Rattus norvegicus Seminiferous Tubules at Stage VIII


title: Seminiferous Tubules Stage (VIII) Replica_5


title: Seminiferous Tubules Stage (VIII) Replica_2


title: Seminiferous Tubules Stage (XIII-XIV) Replica_1


title: Seminiferous Tubules Stage (XII) Replica_2


title: Seminiferous Tubules Stage (IX-XI) Replica_5


title: Seminiferous Tubules Stage (VIIc.d) Replica_1

source_name: Rattus norvegicus Seminiferous Tubules at Stage VIIc.d


title: Seminiferous Tubules Stage (IX-XI) Replica_4


title: Seminiferous Tubules Stage (IX-XI) Replica_3


title: Seminiferous Tubules Stage (IX-XI) Replica_1


title: Seminiferous Tubules Stage (XII) Replica_5


title: Seminiferous Tubules Stage (VIIa.b) Replica_5

source_name: Rattus norvegicus Seminiferous Tubules at Stage VIIa.b


title: Seminiferous Tubules Stage (VIII) Replica_3


title: Seminiferous Tubules Stage (VIII) Replica_1


title: Seminiferous Tubules Stage (VIIa.b) Replica_2


title: Seminiferous Tubules Stage (VI) Replica_3

source_name: Rattus norvegicus Seminiferous Tubules at Stage VI


title: Seminiferous Tubules Stage (VIIc.d) Replica_3


title: Seminiferous Tubules Stage (VIIa.b) Replica_3


title: Seminiferous Tubules Stage (VIIc.d) Replica_5


title: Seminiferous Tubules Stage (VI) Replica_5


title: Seminiferous Tubules Stage (IV-V) Replica_4

source_name: Rattus norvegicus Seminiferous Tubules at Stages IV-V


title: Seminiferous Tubules Stage (VIIc.d) Replica_4


title: Seminiferous Tubules Stage (VIIc.d) Replica_2


title: Seminiferous Tubules Stage (VIIa.b) Replica_4


title: Seminiferous Tubules Stage (VI) Replica_1


title: Seminiferous Tubules Stage (VI) Replica_2


title: Seminiferous Tubules Stage (VIIa.b) Replica_1


title: Seminiferous Tubules Stage (IV-V) Replica_2


title: Mouse_Cerebellum_P2_Ts3

city: Bologna

first: Elisabetta

last: Ciani

address: P.zza Porta San Donato, 2

department: Human and General Physiology

institute: University of Bologna

description: Small focused oligo array containing 112 genes related with cell cycle and 5 "housekeeping" genes.

hyb_protocol: 3 micrograms of biotinilated probe were hybridized overnight at 60°C in GEAhyb Hybridization Solution (SuperArray). Membranes were then washed 1 time at 60°C for 15 min in Wash Solution 1 (2X SSC, 1% SDS) and 1 time at 60°C for 15 min in Wash Solution 2 (0.1X SSC, 0.5% SDS).

scan_protocol: Chemiluminescent detection: Membranes were blocked in GEAblocking Solution Q (SuperArray) for 40 min. Binding of alkaline phosphatase-conjugated streptavidin (SuperArray; diluition 1:8000) was performed for 10 min at room temperature in 1X buffer F (SuperArray). Membranes were then washed 4 times for 5 min with 1X buffer F and rinsed 3 times with buffer G (SuperArray). Membranes were then incubated with CDP-Star chemiluminescent substrate (SuperArray) for 5 min and exposed to X-ray films. Films were then digitalized with a flatbed desktop scanner. Raw intensity value for each spot was evaluated with GEArray Expression Analysis Suite software (SuperArray).

data_processing: Raw intensity values for each spot were background subtracted with mean intensity values from empty (blank) and PUC18 plasmid spots on the array. Background subtracted intensity value for each spot was then normalized to the value of background subtracted intensity of the internal control gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

source_name: Mouse Cerebellum postnatal day 2

label_protocol: Probes were synthesized with TrueLabeling-AMP kit (SuperArray) and purified with ArrayGrade cRNA Cleanup kit (SuperArray).

extract_protocol: Homogenization and RNA isolation with Triazol (Invitrogen). Further purification with Total RNA Isolation Kit (SuperArray).

tag: Strain

value: B6EiC3Sn-T(1716)65Dn (Down syndrome mouse model)

tag: Age

value: postnatal day 2 (P2)

tag: Tissue

value: cerebellum


title: Mouse_Cerebellum_P2_Ts2


title: Seminiferous Tubules Stage (II-III) Replica_4

source_name: Rattus norvegicus Seminiferous Tubules at Stages II-III


title: Seminiferous Tubules Stage (II-III) Replica_3


title: Seminiferous Tubules Stage (IV-V) Replica_5


title: Mouse_Cerebellum_P2_C2

value: B6EiC3Sn, control littermates for Ts65Dn mice (B6EiC3Sn-T(1716)65Dn) Age:postnatal day 2 (P2) Tissue:cerebellum


title: Mouse_Cerebellum_P2_Ts1


title: Seminiferous Tubules Stage I Replica_1

source_name: Rattus norvegicus Seminiferous Tubules at Stage I


title: Mouse_Cerebellum_P2_C3


title: NASH-3

city: Little rock

first: Kartik

last: Shankar

address: 1120 Marshall Street, Slot 512-20B

department: Pharmacology and Toxicology

institute: University of Arkansas for Medical Sciences/Arkansas Childrens Nutrition Center

description: Control-1, 5% Corn Oil

hyb_protocol: Standard Affymetrix protocol

scan_protocol: Standard Affymetrix protocol

data_processing: MAS 5.0 normalization

label_protocol: Standard Affymetrix protocol

extract_protocol: Total RNA was extracted using TRI reagent. RNA was cleaned using Qiagen RNeasy mini columns

treatment_protocol: Rats were fed either 5% Corn oil diet or 70% corn oil diet for 3 weeks via total enteral nutrition

tag: Rat ID 604, Sprague Dawley, male, 225-350 g, Liver


title: Seminiferous Tubules Stage (IV-V) Replica_3


title: Seminiferous Tubules Stage (II-III) Replica_5


title: Seminiferous Tubules Stage I Replica_3


title: Mouse_Cerebellum_P2_C1

data_processing: Raw intensity values for each spot were background subtracted with  mean intensity values from empty (blank) and PUC18 plasmid spots on the array. Background subtracted intensity value for each spot was then normalized to the value of background subtracted intensity of the internal control gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

value: B6EiC3Sn, control littermates for Ts65Dn mice (B6EiC3Sn-T(1716)65Dn)


title: Seminiferous Tubules Stage I Replica_4


title: Seminiferous Tubules Stage (VI) Replica_4


title: Seminiferous Tubules Stage I Replica_2


title: Control-1

description: Control-1, 5% Corn Oil

tag: Sprague Dawley rats, 225-350 g, liver at 3 weeks of diet infusuion


title: Seminiferous Tubules Stage (II-III) Replica_1


title: Control-2

description: Control-1, 5% Corn Oil

tag: Sample ID 616-A, Liver, Sprague-dawley rat, 225-350 g,


title: Seminiferous Tubules Stage I Replica_5


title: Seminiferous Tubules Stage (II-III) Replica_2


title: NASH-1

description: Control-1, 5% Corn Oil

tag: Rat ID 534, Sprague dawley, male, 225-350 g, liver


title: Control-3

description: Control-1, 5% Corn Oil

tag: Rat ID 421, Sprague Dawley, 225-350 g, Liver


title: NASH-2

description: Control-1, 5% Corn Oil

tag: Rat ID 547, Sprague Dawley male, 225-350 g, liver