title: D_NoCysExpF4vsCysExpF1

city: Amsterdam

first: Douwe

last: Molenaar

address: De Boelelaan 1085

department: Systems Bioinformatics

country: Netherlands

institute: Vrije Universiteit Amsterdam

hyb_protocol: Two individual differentially  labeled cDNAs were  incubated at 95° C for 3'  cooled down to 68° C and  mixed (final-volume 20 µl).  To these mixed cDNAs  180 µl of pre-heated (68°  C) Slidehyb#1 hybridization  buffer (Ambion Austin  USA) was added and the  resulting solution was  applied on a pre-heated  slide (68° C). Slides were  then hybridized at 44° C for  16 hours. Subsequently  slides were washed at  42°C once in 1 x  SSC/0.2% SDS and twice  in 1 x SSC and dried by  centrifugation (1 x SSC is  0.15 M NaCl and 0.15 M  Sodium Citrate).

scan_protocol: Using the Scan Array  Express microarray  scanner. 1.Turn on the  scanner and the computer  (in this order) 2.Login:  scanner 3.Double click the  'ScanArray Express' icon  4.Switch on lasers 1 and 3  at least 15 minutes prior to  scanning arrays 5.Click the  'File' button This is to  prevent you from  accidentally turning off the  laser after the 15 minutes  warming-up phase 6.Insert  the slide with the array-side  up and the label towards  the outside. On Agilent  slides the array side is the  side with the label that has  the word Agilent"" on it  7.Press 'Scan | Prescan'  8.Put resolution at 50 m  select both labels used  and set PMT values for  both channels 9.Press  'Start' 10.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 11.Adjust PMT  values to balance signals  obtained for both  channels. If necessary do  a few low-resolution scans  to obtain a optimal balance  12.Click the 'Scan' button  again 13.Select frame for  high resolution scan adjust  resolution (to 10 m) and  scan the array for both  dyes 14.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 15.Switch  off the lasers

data_processing: Microarrays 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).  Statistical significance was  determined from variation  in biological duplicates by  implementation of the  eBayes function included  in Limma (cross-probe  variance estimation) and  false discovery rate  adjustment of the p-values.  In order to characterize the  response of L. reuteri to  glycerol, comparisons were  established (i) during  exponential growth phase  (mid-logarithmic phase  response), (ii) during early  stationary growth phase  (early-stationary phase  response), and (iii) by  comparing the differences  between early-stationary  and mid-logarithmic cells  cultured with and without  glycerol (phase shift  response). Only transcripts  with an average absolute  log2 (ratio) greater than  0.585 and a p-value  smaller than 0.05 were  considered for analysis.  The three lists of  differentially regulated  genes were projected on  metabolic maps of the  genome-scale model  developed in this study.

organism: Limosilactobacillus reuteri subsp. reuteri F275

label_protocol: Annealing Mix x µl RNA  preparation (=5µg) 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’ 70°C  3. cool at room  temperature for 10’  (annealing) 4. spin  down the mixture to the  bottom of the tube 5.  place on ice Reverse  transcription Reverse  Transcription Mix 4 µl 5x  buffer 2 µl 0.1 M DTT 1  µl dNTP-mix 1 µl  AA-dUTP 1 µl TMIII  (keep only briefly  outside 20°C) 20 µl  TOTAL 1. Mix by very  gently by pipetting up  and down (vigorous  pipetting will denature  the enzyme!) 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 µl 2.5  M NaOH 2. Mix (vortex)  and spin down 3.  Incubate for 15 min. at  37°C 4. Add 10 µl 2 M  HEPES free acid 5. Mix  (vortex) and spin down  6. Ready for purification  or store at 20°C  Labelling and  purification of cDNA  with cyanine dyes  Followed Ge Healthcare  kit "Amersham CyDye  (Cy3/Cy5)Reactive Dye  Protocols for Post  Labelling  Aminoallyl-cDNA and  Aminoallyl-aRNA" code  RPN5661 Labelling of  amino allyl-modified  cDNA with CyDye 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  CyDye-labelled cDNA  Purification of CyDye  labelled cDNA Followed  Ge Healthcare kit  "Amersham CyDye  (Cy3/Cy5)Reactive Dye  Protocols for Post  Labelling  Aminoallyl-cDNA

extract_protocol: Collecting cells  Quenching Buffer 66.7  mM HEPES (pH 6.5)  60%Methanol Extraction  Mixture (Prepared in a  screw-cap tube) -500 µll  phenol/Chloroform -30  µl10% SDS -30 µl3 M  NaAc (pH 5.2) -500 mg  glass-beads (75-150  µm) -400 µlTE buffer  1.For each sample  prepare a tube with  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) 5.Discard the  supernatant; keep the  cell pellet cooled and  work quickly to prevent  condensation of water.  Transfer the pellet with  a pre-cooled spatula to  a screw-crap tube with  Extraction Mixture 6.Mix  the cell pellet thoroughly  with Extraction Mixture  by shaking by hand  7.Freeze the tube  immediately in liquid  nitrogen 8.Break cells in  the Savant FastPrep  FP120. Three times 40  seconds at speed 4.0  (cool cells between  steps if necessary)  9.Centrifuge for 5  minutes at 4°C in an  Eppendorf centrifuge at  10.000 rpm 10.Transfer  500 µl of the  supernatant to a new  tube. Add 400 µl  chloroform (chloroform  should be cooled at  4°C) 11.Centrifuge for 2  minutes in an Eppendorf  centrifuge at full speed  (4°C) 5.Continue with  the "High Pure RNA  Isolation Kit" from  Roche (order # 1 828  665) 6.Incubate for 60  minutes with DnaseI,  elute with 50 µl elution  buffer 7.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.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-244B

status: Public on Oct 31 2008

type: RNA

city: Bethesda

first: Xin

middle: Wei

last: Wang

state: MD

address: 37 Convent Drive

department: Laboratory of Human Carcinogenesis

country: USA

institute: National Cancer Institute

description: No additional information.

hyb_protocol: Sample Hybridization Protocol Qty. of labeled target: 5 ug of total RNA Other: Labeled targets from 5ug of total RNA were used for hybridization on each miRNA microarray chip containing human and mouse miRNA genes. All probes on these microarrays are 40-mer oligonucleotides spotted by contacting technologies and covalently attached to a polymeric matrix. The microarrays were hybridized in 6x SSPE (0.9 M NaCl / 60 mM NaH2PO4 H20 / 8 mM EDTA, pH 7.4) / 30% formamide at 25C for 18 hr, washed in 0.75x TNT (Tris HCL/NaCl/Tween 20) at 37C for 40 min.

scan_protocol: Scanning Protocol Other: Processed slides were scanned by using a PerkinElmer ScanArray XL5K Scanner, with the laser set to 635 nm, at Power 80 and PMT 70 setting, and a scan resolution of 10 um.

data_processing:
Data Processing Protocol
Calculation Method: Raw data were normalized and analyzed in GENESPRING software version 6.1.1 (Silicon Genetics, Redwood City, CA). GENESPRING generated an average value of the spot replicates of each miRNA. After data transformation (to convert any negative value to 0.01), normalization was performed by using a per-chip on median normalization and a normalization to specific samples used as common reference for miRNA expression.

label: alexa 647

molecule: total RNA

organism: Homo sapiens

label_protocol:
Labeling Protocol
Other: Processed by using a method of direct detection of the biotin-containing transcripts by streptavidin-Alexa647 conjugate.

extract_protocol:
Tri-Reagent Protocol
Other: Total RNA isolation was performed using the Tri-Reagent Protocol (Molecular Research Center, Cincinnati)

tag: Gender

value: male

tag: Tissue

value: liver tissue (non-tumorous)

tag: Disease state

tag: Individual

tag: Clinical info

value: Follow up status:dead


title: Hepatocellular carcinoma (HCC), mestasis free, 03-256B

description: No additional information.

value: Follow up status:alive


title: Hepatocellular carcinoma (HCC), mestasis free, 03-263B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-261B

description: No additional information.


title: E_CysEarlyStaF3vsNoCysEarlyStaF4


title: P_NoCysExpF4vsNoCysEarlyStaF4


title: Hepatocellular carcinoma (HCC), mestasis free, 03-241B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-235B

description: No additional information.


title: G_CysExpF1vsCysExpF3


title: Hepatocellular carcinoma (HCC), mestasis free, 03-234B

description: No additional information.

value: female


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-243B

description: No additional information.

value: Follow up status:


title: K_NoCysEarlyStaF4vsCysEarlyStaF3


title: N_NoCysExpF2vsNoCysExpF2


title: B_NoCysExpF2vsCysExpF3


title: F_NoCysEarlyStaF4vsCysExpF1


title: H_CysExpF3vsCysEarlyStaF3


title: Hepatocellular carcinoma (HCC), mestasis free, 03-233B

description: No additional information.


title: J_CysEarlyStaF3vsNoCysExpF2


title: C_CysExpF3vsNoCysExpF4


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-227B

description: No additional information.


title: O_CysEarlyStaF3vsCysExpF3


title: Hepatocellular carcinoma (HCC), mestasis free, 03-231B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-228B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-226B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-237B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-230B

description: No additional information.


title: A_CysExpF1vaNoCysExpF2 #2

description: A_CysExpF1vaNoCysExpF2 #2


title: Hepatocellular carcinoma (HCC), mestasis free, 03-219B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-223B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-214B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-215B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-218B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-225B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-205B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-211B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-199B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-182B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-200B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-208B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-195B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-187B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-165B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-220B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-197B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-150B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-210B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-149B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-162B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-164B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-196B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-192B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-151B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-119B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-105B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-180B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-146B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-093B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-112B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-139B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-098B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-148B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-089B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-147B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-081B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-138B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-087B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-133B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-116B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-102B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-088B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-082B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-062B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-071B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-064B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-050B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-059B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-055B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-077B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-061B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-054B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-070B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-048B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-058B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-057B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-049B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-043B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-025B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-051B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-047B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-037B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-455B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-017B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-012B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-033B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-429B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 03-023B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-042B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-454B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-461B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-024B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-445B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-431B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-459B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-466B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-447B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-452B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-456B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-458B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-432B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-460B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-073B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-448B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-457B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 03-015B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-430B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-424B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-423B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-421B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-416B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-409B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-404B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-415B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-407B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-345B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-396B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-342B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-362B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-355B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-361B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-398B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-370B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-344B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-339B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-363B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-395B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-366B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-347B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-375B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-354B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-387B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-346B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-384B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-358B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-325B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-337B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-323B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-314B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-318B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-315B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-313B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-321B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-298B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-286B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-289B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-290B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-295B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-302B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-287B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-301B

description: No additional information.


title: Hepatocellular carcinoma (HCC), venous mestasis, 02-307B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-309B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-269B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-291B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-285B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-271B

description: No additional information.


title: Hepatocellular carcinoma (HCC), mestasis free, 02-283B

description: No additional information.


title: S_GluExpF2pNZ7748vsGluStaF2pNZ7748

hyb_protocol: Two individual differentially , labeled cDNAs were , incubated at 95° C for 3' , cooled down to 68° C and , mixed (final-volume 20 µl). , To these mixed cDNAs , 180 µl of pre-heated (68° , C) Slidehyb#1 hybridization , buffer (Ambion Austin , USA) was added and the , resulting solution was , applied on a pre-heated , slide (68° C). Slides were , then hybridized at 44° C for , 16 hours. Subsequently , slides were washed at , 42°C once in 1 x , SSC/0.2% SDS and twice , in 1 x SSC and dried by , centrifugation (1 x SSC is , 0.15 M NaCl and 0.15 M , Sodium Citrate).

scan_protocol: Using the Scan Array ,   Express microarray ,   scanner. 1.Turn on the ,   scanner and the computer   , (in this order) 2.Login: ,   scanner 3.Double click the   , 'ScanArray Express' icon   , 4.Switch on lasers 1 and   3 , at least 15 minutes  prior  to , scanning arrays  5.Click  the , 'File' button  This is to  , prevent you  from ,  accidentally turning  off the  , laser after the 15  minutes  , warming-up  phase  6.Insert , the slide  with the  array-side , up  and the  label towards ,  the outside.  On Agilent ,  slides the  array side is the  , side with  the label that  has , the  word  Agilent""  on it ,  7.Press 'Scan |  Prescan' ,  8.Put resolution  at 50 m ,  select both  labels used ,  and set PMT  values for ,  both channels  9.Press ,  'Start' 10.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 11.Adjust PMT ,   values to balance signals ,   obtained for both ,   channels. If necessary do ,   a few low-resolution scans   , to obtain a optimal   balance , 12.Click the   'Scan' button , again   13.Select frame for , high   resolution scan adjust ,   resolution (to 10 m) and ,   scan the array for both ,   dyes 14.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 , 15.Switch off the   lasers

data_processing: the VALUE column is calculated as log2 of ratio (SIGNAL_CH1/SIGNAL_CH2). SIGNAL_CH1 is calculated as F_CH1_MEAN - B_CH1_MEAN and SIGNAL_CH2 is calculated as F_CH2_MEAN - B_CH2_MEAN.


title: O_GluGlyExpF4pNZ7748vsGluGlyStaF4pNZ7748


title: R_GluGlyStaF4pNZ7748vsGluStaF6pNZ7748


title: P_GluGlyStaF3pNZ7021vsGluGlyExpF3pNZ7021


title: N_GluGlyExpF4pNZ7748vsGluExpF2pNZ7748


title: Q_GluStaF2pNZ7748vsGluGlyStaF8pNZ7748


title: I_GluStaF2pNZ7748vsGluStaF1pNZ7021


title: F_GluGlyStaF8pNZ7748vsGluStaF6pNZ7748


title: M_GluExpF2pNZ7748vsGluExpF1pNZ7021


title: L_GluExpF1pNZ7021vsGluGlyExpF3pNZ7021


title: Hepatocellular carcinoma (HCC), mestasis free, 02-284B

description: No additional information.


title: J_GluGlyStaF4pNZ7748vsGluStaF2pNZ7748


title: K_GluGlyExpF3pNZ7021vsGluGlyExpF4pNZ7748


title: Spleen-T Treg_biological_rep3

city: St. Louis

first: Yu

last: Zhao

state: MO

address: 660 S. Euclid

institute: Washington University

hyb_protocol: Chips hybridized according to Manufacturer's protocol

data_processing: MAS5 signal

label: biotin

organism: Mus musculus

source_name: IP-tumor Spleen Treg

label_protocol: Chips labeled with biotin according to Manufacturer's protocol

extract_protocol:
Cell isolation: Naïve Treg cells were purified from the resting spleens of FoxP3-GFP knock-in mice made in the 129/SvJ strain. Ascites Treg cells were tumor-associated FoxP3-GFP+ cells sorted from the tumor ascites of 129/SvJ mice IP-injected with 1 x 10e6 RMAS cells 5 days earlier; Spleen-T Treg cells were purified from the spleens of the same tumor-bearing mice.
RNA extracted with Trizol

tag: Genotype

value: Foxp3-GFP KI, Spleen-T Treg cells were purified from the spleens of the same tumor-bearing mice.


title: G_GluGlyStaF3pNZ7021vsGluGlyStaF4pNZ7748


title: E_GluStaF6pNZ7748vsGluStaF5pNZ7021


title: D_GluStaF5pNZ7021vsGluGlyStaF7pNZ7021


title: H_GluStaF1pNZ7021vsGluGlyStaF3pNZ7021


title: Spleen-T Treg_biological_rep1


title: C_GluGlyStaF7pNZ7021vsGluGlyStaF8pNZ7748


title: Spleen-T Treg_biological_rep2


title: Afghan Hound

type: genomic

city: Seattle

first: Thomas

middle: J

last: Nicholas

state: WA

address: 1705 NE Pacific St. Foege Building

department: Genome Sciences

institute: University of Washington

description: Hybridization of Afghan Hound test sample and Boxer reference sample

hyb_protocol: Performed by Nimblegen standard protocol

data_processing: Normalized by standard Nimblegen methods.

molecule: genomic DNA

organism: Canis lupus familiaris

source_name: Afghan Hound

label_protocol: Labeled by Nimblegen

extract_protocol: Qiagen QIAamp DNA Blood Maxi Kit

tag: Male Afghan Hound sample

source_name: Boxer

tag: Female Boxer sample


title: Foxa2-mutant-2-E7.5 (U74)

city: Madison

first: Owen

middle: James

last: Tamplin

state: WI

address: 1111 Highland Ave

department: Cell and Regenerative Biology

institute: University of Wisconsin-Madison

description:
E7.5 POOLED EMBRYOS: n=48
Stages between mid-streak and headfold according to Downs & Davies

hyb_protocol: Affymetrix

scan_protocol: Affymetrix

data_processing: Affymetrix MAS5.0, target normalized to 1000

source_name: E7.5 EMBRYO EPIBLAST ONLY

label_protocol: AFFYMETRIX GeneChip® T7-Oligo(dT) Promoter Primer Kit

extract_protocol: TRIZOL REAGENT

treatment_protocol: EXTRA-EMBRYONIC REGION OF EMBRYO REMOVED PRIOR TO EXTRACTION

tag: Embryos derived by tetraploid complementation technique using Foxa2 homozygous null ES cells.


title: Basenji

description: Hybridization of Basenji test sample and Boxer reference sample

scan_protocol: Performed by Nimblegen standard protocol

source_name: Basenji

tag: Female Basenji sample


title: Belgian Shepherd Dog Tervuren

description: Hybridization of Belgian Shepherd Dog Tervuren test sample and Boxer reference sample

source_name: Belgian Shepherd Dog Tervuren

tag: Male Belgian Shepherd Dog Tervuren sample


title: Foxa2-mutant-1-E7.5 (U74)

description:
E7.5 POOLED EMBRYOS: n=53
Stages between mid-streak and headfold according to Downs & Davies


title: Wild type 2 E7.5 (U74)

description:
E7.5 POOLED EMBRYOS: n=50
Stages between mid-streak and headfold according to Downs & Davies

tag: STRAIN

value: ICR

tag: GENDER

value: MIXED


title: Wild type 1 E7.5 (U74)


title: Thalamus, MPTP day 12, replicate 5

status: Public on Oct 30 2008

city: Kuopio

first: Markus

last: Storvik

address: University of Eastern Finland

department: Pharmacology

country: Finland

institute: University of Eastern Finland

description: Thalamus, MPTP day 12, replicate 5, raw data

hyb_protocol: According to the manufacturer's instructions.

scan_protocol: At 3-Am resolution using the GeneChip System confocal scanner (Hewlett-Packard, Santa Clara, CA) with a 488-nm emission and detection at 70 nm, controlled by Affymetrix Microarray Suite 5.0 (Affymetrix).

organism: Macaca fascicularis

source_name: MPTP treated macaque, day 12, replicate 5

label_protocol: According to the standard protocol outlined in the Affymetrix GeneChip Expression Analysis Technical Manual

growth_protocol: Animals were housed in individual primate cages under controlled conditions of humidity (50 F 5%), temperature (24 F 18C) and light (12h light/12h dark cycles, time lights on 8:00 am), food and water were available ad libitum and animal care was supervised by veterinarians skilled in the healthcare and maintenance of nonhuman primates. Experiments were carried out in accordance with European Communities Council Directive of 24 November 1986 (86/609/EEC) for care of laboratory animals.

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

treatment_protocol: Treated daily (9:00 am) with MPTP hydrochloride (0.2 mg/kg, i.v., Sigma, St. Louis, MO) dissolved in saline. Sacrificed in ketamine anesthesia by sodium pentobarbital overdose

tag: State

value: Presymptomatic

value: Thalamus


title: Thalamus, Saline, replicate 2

description: Saline treated macaque, replicate 2, raw data

source_name: Saline treated macaque, replicate 2

value: Saline


title: Rottweiler

description: Hybridization of Rottweiler test sample and Boxer reference sample

source_name: Rottweiler

tag: Female Rottweiler sample


title: Thalamus, Saline, replicate 3

description: Saline treated macaque, replicate 3, raw data

source_name: Saline treated macaque, replicate 3


title: Thalamus, Saline, replicate 1

description: Saline treated macaque, replicate 1, raw data

source_name: Saline treated macaque, replicate 1


title: Golden Retriever

description: Hybridization of Golden Retriever test sample and Boxer reference sample

source_name: Golden Retriever

tag: Male Golden Retriever sample


title: Thalamus, MPTP day 12, replicate 4

description: Thalamus, MPTP day 12, replicate 4, raw data

source_name: MPTP treated macaque, day 12, replicate 4


title: Thalamus, Saline, replicate 4

description: Saline treated macaque, replicate 4, raw data

source_name: Saline treated macaque, replicate 4


title: German Shorthaired Pointer

description: Hybridization of German Shorthaired Pointer test sample and Boxer reference sample

source_name: German Shorthaired Pointer

tag: Male German Shorthaired Pointer sample


title: Doberman Pinscher

description: Hybridization of Doberman Pinscher test sample and Boxer reference sample

source_name: Doberman Pinscher

tag: Male Doberman Pinscher sample


title: Shetland Sheepdog

description: Hybridization of Shetland Sheepdog test sample and Boxer reference sample

source_name: Shetland Sheepdog

tag: Male Shetland Sheepdog


title: Boxer (Tasha)

description: Hybridization of Boxer (Tasha) test sample and Boxer reference sample

source_name: Boxer (Tasha)

tag: Female Boxer (Tasha) sample


title: Whippet

description: Hybridization of Whippet test sample and Boxer reference sample

source_name: Whippet

tag: Male Whippet sample


title: Pug

description: Hybridization of Pug test sample and Boxer reference sample

source_name: Pug

tag: Female Pug sample

source_name: Boxer


title: set2 null(BY4741) (Cy5) Total RNA vs. Wt(BY4741) (Cy3) Total RNA #2

city: Chapel Hill

first: Jason

middle: D

last: Lieb

state: NC

address: 408 Fordham Hal, CB#3280

department: Biology

institute: UNC-Chapel Hill

description: Wild type (BY4741) and set2∆ (BY4741) strains were grown at 30°C in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD600 of 0.6-0.8. For each of the three replicates, Total RNA was extracted by acid-phenol method (Xiao et al. 2003). Double-stranded cDNA was prepared using an Invitrogen SuperScript™ (Cat No. 11917-010) primed with Oligo(dt) and random hexamers.

hyb_protocol: Samples were hybridized by Nimblegen Systems, Inc. Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nat Biotechnol. 1999 Oct;17(10):974-8. PMID: 10504697 Performed by Nimblegen: http://www.nimblegen.com/products/lit/lit.html

scan_protocol: Samples were scanned by Nimblegen Systems, Inc. Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nat Biotechnol. 1999 Oct;17(10):974-8. PMID: 10504697 Performed by Nimblegen: http://www.nimblegen.com/products/lit/lit.html

data_processing:
Array Scans were analyzed by Nimblegen Inc. using Genepix Software
Data was normalized by median centering log2 ratios
Performed by Nimblegen:
http://www.nimblegen.com/products/lit/lit.html

organism: Saccharomyces cerevisiae

source_name: Total RNA from WT(BY4741) Mid Log Growth

extract_protocol:
Total RNA is Extracted by the acid-phenol method:
Xiao, T., Hall, H., Kizer, K.O., Shibata, Y., Hall, M.C., Borchers, C.H., and Strahl, B.D. 2003. Phosphorylation of RNA polymerase II CTD regulates H3 methylation in yeast. Genes and Development 17(5): 654-663.
Double-stranded cDNA was prepared using an Invitrogen SuperScript™ (Cat No. 11917-010) primed with Oligo(dt) and random hexamers

source_name: Total RNA from set2 null(BY4741) Mid Log Growth

tag: set2 null(BY4741)


title: German Shepherd

description: Hybridization of German Shepherd test sample and Boxer reference sample

source_name: German Shepherd

tag: Female German Shepherd sample


title: Thalamus, MPTP day 12, replicate 3

description: Thalamus, MPTP day 12, replicate 3

source_name: Thalamus, MPTP day 12, replicate 3

tag: Tissue


title: Thalamus, MPTP day 6, replicate 4

description: Thalamus, MPTP day 6, replicate 4

source_name: Thalamus, MPTP day 6, replicate 4


title: West Highland White Terrier

description: Hybridization of West Highland White Terrier test sample and Boxer reference sample

source_name: West Highland White Terrier

tag: Female West Highland White Terrier sample

tag: Female Boxer sample


title: Thalamus, MPTP day 6, replicate 3

description: Thalamus, MPTP day 6, replicate 3

source_name: Thalamus, MPTP day 6, replicate 3


title: set2 null(BY4741) (Cy3) Total RNA vs. Wt(BY4741) (Cy5) Total RNA

source_name: Total RNA from set2 null (BY4741) Mid Log Growth

label_protocol:
Performed by Nimblegen:
http://www.nimblegen.com/products/lit/lit.html

tag: set2 null


title: Gray Wolf

description: Hybridization of Gray Wolf test sample and Boxer reference sample

organism: Canis lupus

source_name: Gray Wolf

tag: Male Gray Wolf sample


title: Siberian Husky

description: Hybridization of Siberian Husky test sample and Boxer reference sample

source_name: Siberian Husky

tag: Female Siberian Husky sample


title: Yorkshire Terrier

description: Hybridization of Yorkshire Terrier test sample and Boxer reference sample

source_name: Yorkshire Terrier

tag: Female Yorkshire Terrier sample


title: Labrador Retriever

description: Hybridization of Labrador Retriever test sample and Boxer reference sample

source_name: Labrador Retriever

tag: Female Labrador Retriever sample


title: Standard Poodle

description: Hybridization of Standard Poodle test sample and Boxer reference sample

source_name: Standard Poodle

tag: Male Standard Poodle sample


title: set2 null(BY4741) (Cy5) Total RNA vs. Wt(BY4741) (Cy3) Total RNA


title: Putamen, MPTP day 25, replicate 5

description: Putamen of MPTP treated macaque, day 25, replicate 5.

source_name: Putamen, MPTP day 25, replicate 5

tag: State

value: Symptomatic

value: Putamen


title: Thalamus, MPTP day 12, replicate 1

description: Thalamus, MPTP day 12, replicate 1

source_name: Thalamus, MPTP day 12, replicate 1


title: Thalamus, MPTP day 6, replicate 1

description: Thalamus, MPTP day 6, replicate 1

source_name: Thalamus, MPTP day 6, replicate 1


title: Thalamus, MPTP day 6, replicate 2

description: Thalamus, MPTP day 6, replicate 2

source_name: Thalamus, MPTP day 6, replicate 2


title: Thalamus, Control, replicate 3

description: Thalamus, Control, replicate 3.

source_name: Thalamus, Control, replicate 3

value: Control


title: Putamen, Saline, replicate 5

description: Putamen of Saline treated macaque, replicate 5.

data_processing: MAS5.0 (mas5)

source_name: Putamen, Saline, replicate 5


title: Thalamus, Controls, replicate 4

description: Thalamus, Control, replicate 4.

source_name: Thalamus, Controls, replicate 4


title: Thalamus, MPTP day 25, replicate 1

description: Thalamus, MPTP day 25, replicate 5

source_name: Thalamus, MPTP day 25, replicate 1


title: Thalamus, MPTP day 25, replicate 4

description: Thalamus, MPTP day 25, replicate 4.

source_name: Thalamus, MPTP day 25, replicate 4


title: Thalamus, Control, replicate 5

description: Thalamus, Control, replicate 5.

source_name: Thalamus, Control, replicate 5


title: Putamen, MPTP day 6, replicate 4

description: Putamen of MPTP treated macaque day 6, replicate 4.

source_name: Putamen, MPTP day 6, replicate 4


title: Thalamus, Control, replicate 1

description: Thalamus, Control, replicate 1.

source_name: Thalamus, Control, replicate 1

value: Controls


title: Putamen, MPTP day 6, replicate 5

description: Putamen of MPTP treated macaque, day 6, replicate 5.

source_name: Putamen, MPTP day 6, replicate 5


title: Putamen, Saline, replicate 4

description: Putamen of Saline treated macaque, replicate 4.

source_name: Putamen, Saline, replicate 4


title: Thalamus, Control, replicate 2

description: Thalamus, Control, replicate 2.

source_name: Thalamus, Control, replicate 2


title: Thalamus, MPTP day 12, replicate 2

description: Thalamus, MPTP day 12, replicate 2

source_name: Thalamus, MPTP day 12, replicate 2


title: Boxer self hybridization

description: Self Hybridization of Boxer test sample and Boxer reference sample

source_name: Boxer


title: Thalamus, MPTP day 25, replicate 3

description: Thalamus, MPTP day 25, replicate 3

source_name: Thalamus, MPTP day 25, replicate 3


title: Putamen, MPTP day 25, replicate 3

description: Putamen of MPTP treated macaque day 25, replicate 3.

source_name: Putamen, MPTP day 25, replicate 3


title: Putamen, MPTP day 6, replicate 3

description: Putamen of MPTP treated macaque day 6, replicate 3.

source_name: Putamen, MPTP day 6, replicate 3


title: Thalamus, MPTP day 25, replicate 2

description: Thalamus, MPTP day 25, replicate 2

source_name: Thalamus, MPTP day 25, replicate 2


title: Putamen, MPTP day 12, replicate 4

description: Putamen of MPTP treated macaque day 12, replicate 4.

source_name: Putamen, MPTP day 12, replicate 4


title: Putamen, Saline, replicate 3

description: Putamen of Saline treated macaque, replicate 3.

source_name: Putamen, Saline, replicate 3


title: GPi Saline replicate 2

description: GPi of Saline treated macaque, replicate 2.

source_name: Saline treated macaque

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

value: Saline control


title: Putamen, MPTP day 25, replicate 4

description: Putamen of MPTP treated macaque day 25, replicate 4.

source_name: Putamen, MPTP day 25, replicate 4


title: Putamen, Saline, replicate 2

description: Putamen of Saline treated macaque, replicate 2.

source_name: Putamen, Saline, replicate 2


title: Putamen, MPTP day 25, replicate 2

description: Putamen of MPTP treated macaque at 25 days, replicate 2.

source_name: Putamen, MPTP day 25, replicate 2


title: GPi MPTP day 6, replicate 2

description: GPi of MPTP treated macaque day 6, replicate 2.

source_name: MPTP treated macaque


title: GPi MPTP day 25, replicate 2

description: GPi of MPTP treated macaque day 25, replicate 2.


title: Putamen, MPTP at day 12, replicate 2

description: Putamen of MPTP treated macaque at 12 days, replicate 2.


title: GPi MPTP day 6 repl 3

description: GPi of MPTP treated macaque day 6, replicate 3.


title: Gpi Saline replicate 4

description: GPi of Saline treated macaque, replicate 4.


title: GPi Saline replicate 3

description: GPi of saline treated macaque, replicate 3.


title: GPi Saline replicate 1

description: GPi of Saline treated macaque, replicate 1.


title: GPi MPTP say 12, replicate 2

description: GPi of MPTP treated macaque day 12, replicate 2.


title: GPi MPTP day 12 replicate 1

description: GPi of MPTP treated macaque day 12, replicate 1.


title: GPi, MPTP treatment day 12, replicate 3

description: GPi of MPTP treated macaque at 6 days, replicate 3.

source_name: GPi MPTP day 12


title: GPi MPTP day 25 replicate 1

description: GPi of MPTP treated macaque day 25, replicate 1.


title: MPTP day 6, replicate 1

description: GPi of MPTP treated macaque day 6, replicate 1.


title: Gpi MPTP day 25, replicate 3

description: GPi of MPTP treated macaque day 25, replicate 3.


title: Msi1cy3_GFPcy5_1_4

city: San Antonio

first: Luiz

last: Penalva

state: TX

address: 8403 Floyd Curl Drive

department: GCCRI

institute: UTHSCSA

description: Msi1-Cy3, GFP-Cy5 slide 1, microarray 4

hyb_protocol: 825ng of Msi1-Cy3 and GFP-Cy5 labeled cRNA were hybridized by the Agilent hybridization protocol for 17 hours at 65°C, 10 rpm in a hybridization oven purchased from Agilent (G2545A). Arrays were washed with Agilent gene expression wash buffers 1 and 2 with 0.005% Triton X-10, as directed by Agilent protocol.

scan_protocol: Arrays were scanned according to the recommended Agilent scanner settings provided in the Agilent two-color microarray based gene expression analysis (quick amp labeling) protocol. The arrays were scanned with a scan area of 61 x 21.6 mm, a scan resolution of 5μm, the 5 μm scanning mode as single pass, extended Dynamic range selected, the dye channels of red and green, the green PMT set for XDR Hi 100% XDR Lo 10%, the red PMT set for XDR Hi 100% XDR Lo 10%.

data_processing: TIFF images were quantified using Agilent Feature Extraction Software 9.5. The raw data was corrected for background intensity, followed by global loess normalization and quantile normalization between arrays using LIMMA package of Bioconductor.

source_name: HEK293T Cells

label_protocol: 200ng of total RNA was reverse transcribed to cDNA using T7 Promoter primer and MMLV-RT. cDNA was converted to cRNA using T7 RNA polymerase and Cy3-CTP, and NTPs using the Agilent two-color microarray based gene expression analysis (quick amp labeling) protocol.

growth_protocol: Grown at 37°C in 5% CO2

extract_protocol: Trizol and RNeasy columns

treatment_protocol: HEK293T cells were transfected wit BAP (Biotin Acceptor Peptide) tagged with Msi1. After 48 hours RNA was purified with Trizol reagent and further purified with RNeasy columns.

tag: Treatment

value: Transient transfection with BAP tagged with Msi1

tag: Time

value: 48 hours

label_protocol: 200ng of total RNA was reverse transcribed to cDNA using T7 Promoter primer and MMLV-RT. cDNA was converted to cRNA using T7 RNA polymerase and Cy5-CTP, and NTPs using the Agilent two-color microarray based gene expression analysis (quick amp labeling) protocol.

treatment_protocol: HEK293T cells were transfected wit BAP (Biotin Acceptor Peptide) tagged with GFP. After 48 hours RNA was purified with Trizol reagent and further purified with RNeasy columns.

value: Transient transfection with BAP tagged with GFP


title: 13279328 - NonPol-Tr-2 vs NonPol-Co-2

status: Public on Oct 29 2008

city: BEAUCOUZE

first: Sandra

last: Pelletier

address: 42, rue Georges Morel - BP 60057

country: France

institute: INRA

description: Transcriptional and translational status of genes responding to drought stress

hyb_protocol: NonPol-Co-2 Cy5 / NonPol-Tr-2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

scan_protocol: GenePix Pro 3.0, Cy3:pmt voltage 532nm,650V,laser power 100%, Cy5:635nm,pmt voltage 620V,laser power 100%

data_processing: The raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter (Flags=-100). Then we performed a global intensity-dependent normalization using the loess procedure (see Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock.

organism: Arabidopsis thaliana

label_protocol: labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).

growth_protocol: aerial - Media : sand + half-strength Hoaglands solution	 hygrometry : 60%	 Temperature : 21°C	 Light : 8 hr light

treatment_protocol: no treatment

tag: Arabidopsis thaliana (col-0)  - dev.stage (Boyes et al. Plant Cell 2001):boyes,boyes 1.06

treatment_protocol: Name:Drought_GEN26 - abiotic stress - compound starvation,water:time 6.5day . Progressive drought stress was applied by withholding watering. The intensity of water-deficit was quantified by measuring of volumetric water content as described (http://jan.ucc.nau.edu/doetqp/courses/env320/lec9/) and as an additional control, by the visual monitor of incipient leaf wilting in the drought-sensitive mutant ost1-2 grown in parallel (Merlot, S. et al. 2002 Plant J. 30, 601-609; Mustilli et al., 2002 Plant Cell 14, 3089-3099). Wild-type rosette leaves from the stressed plants were harvested at stage 1.06-1.08 according to Boyes et al. (2001) when the ost1-2 leaves showed loss of turgescence (volumetric water content = 7 %). As control references, rosette leaves were collected in parallel from well-watered plants.


title: GFPcy3_Msi1cy5_2_4

description: GFP-Cy3, Msi1-Cy5 slide 2, microarray 4

hyb_protocol: 825ng of GFP-Cy3 and Msi1-Cy5 labeled cRNA were hybridized by the Agilent hybridization protocol for 17 hours at 65°C, 10 rpm in a hybridization oven purchased from Agilent (G2545A). Arrays were washed with Agilent gene expression wash buffers 1 and 2 with 0.005% Triton X-10, as directed by Agilent protocol.

value: Transient transfection with BAP tagged with GFP;

value: Transient transfection with BAP tagged with Msi1;


title: Msi1cy3_GFPcy5_2_3

description: Msi1-Cy3, GFP-Cy5 slide 2, microarray 3


title: Msi1cy3_GFPcy5_2_1

description: Msi1-Cy3, GFP-Cy5 slide 2, microarray 1


title: GFPcy3_Msi1cy5_1_3

description: GFP-Cy3, Msi1-Cy5 slide 1, microarray 3


title: 13279320 - Pol-Tr-2 vs Pol-Co-2

hyb_protocol: Pol-Co-2 Cy5 / Pol-Tr-2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: GFPcy3_Msi1cy5_1_1

description: GFP-Cy3, Msi1-Cy5 slide 1, microarray 1


title: GFPcy3_Msi1cy5_2_2

description: GFP-Cy3, Msi1-Cy5 slide 2, microarray 2


title: 13279330 - NonPol-Co-2 vs NonPol-Tr-2

hyb_protocol: NonPol-Tr-2 Cy5 / NonPol-Co-2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13278984 - Pol-Tr-1 vs Pol-Co-1

hyb_protocol: Pol-Co-1 Cy5 / Pol-Tr-1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13281172 - Tot-Tr-2 vs Tot-Co-2

hyb_protocol: Tot-Co-2 Cy5 / Tot-Tr-2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13279318 - Tot-Co-2 vs Tot-Tr-2

hyb_protocol: Tot-Tr-2 Cy5 / Tot-Co-2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13279324 - Pol-Co-2 vs Pol-Tr-2

hyb_protocol: Pol-Tr-2 Cy5 / Pol-Co-2 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Msi1cy3_GFPcy5_1_2

description: Msi1-Cy3, GFP-Cy5 slide 1, microarray 2


title: 13279321 - Pol-Co-1 vs Pol-Tr-1

hyb_protocol: Pol-Tr-1 Cy5 / Pol-Co-1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13281043 - Tot-Tr-1 vs Tot-Co-1

hyb_protocol: Tot-Co-1 Cy5 / Tot-Tr-1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13278971 - Tot-Co-1 vs Tot-Tr-1

hyb_protocol: Tot-Tr-1 Cy5 / Tot-Co-1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 13279307 - NonPol-Co-1 vs NonPol-Tr-1

hyb_protocol: NonPol-Tr-1 Cy5 / NonPol-Co-1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Rice moderate drought Rice28

city: Golm

first: Karin

last: Koehl

state: Brandenburg

address: Am Muehlenberg 1

country: Germany

institute: Max-Planck-Institut of Molecular Plant Physiology

description: no additional description

hyb_protocol: Prehybridization was done following the manufactures instructions: blocking of slide for 20 min at 42°C with Block Solution (2X SSC, 0.05% SDS, 0.25% NaBH4) followed by washing of slide with 1X SSC, 0.2X SSC and rinsing with Nanopure water. For hybridization labelled DNA was dissolved in 130µl hybridization buffer (0.1 % SDS, 25 % Formamide, 5x SSC, 10 mg/ml BSA, 40 mM Na3PO4 (pH 6.8)) and applied to the pre-warmed slide in the hybridization station Hybarray12 (Perkin Elmer, Wellesley, MA, USA). The Initial hybridization temperature was 45°C. The hybridization temperature was stepwise reduced every hour by 1K to reach final hybridization temperature of 41°C. After 10-12 hours of hybridization at 41°C, slides were washed with 2X SSC, 0.2% SDS at 42°C, 2X SSC and 0.2X SSC (all 4 cycles with 20s flow and 40s hold) and very shortly in Nanopure water, all at room temperature. Slides were dried in a centrifuge.

scan_protocol: Microarrays were scanned with a FLA-8000 laser scanner (Fuji, Tokyo, Japan) at maximum intensity. Software version was FLA-8000 V 1.12.

data_processing: The software GeneSpotter 2.4.3 (Microdiscovery, Berlin, Germany) was used to fit the grid position of the spots and to calculate spot intensities. Spots for which intensity was affected by dust or dirt were flagged manually (user tag column = 1). Background correction was done based on the expression signal of 218 spots representing a hygromycin resistance gene that is not in the genome of the investigated cultivars. Any spot with an expression below the mean plus threefold standard deviation of the intensity of the hygromycin gene spots of the respective array and dye was labelled as below background (tag column = 1). Normalization was performed using the R package limma (R 2.3.1, limma version 2.7.3; (Smyth, 2005)). The methods Robustspline for within array normalization and Quantile for between array normalization were applied.

label: Alexa Fluor 532

molecule: polyA RNA

organism: Oryza sativa

source_name: rice leaf;IR 57311;control

label_protocol: For labeling 1 mg cDNA (in 15 µl buffer (10 mM Tris, pH 8.5)) were mixed with 5 µl of the fluorescent dye (Alexa Fluor 532 and 647 (Qiagen, Hilden, Germany)) and incubated for 30 - 45 min at 85 °C in the dark, followed by adding of 5 µl Stop Solution and an incubation for 10 min at room temperature.

growth_protocol: Seeds were pre-germinated in tab water at 28°C for ten days. Then, plantlets were transferred in 10 cm diameter pots filled with 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany), an ion-exchange resin loaded with nutrient ions and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were transferred to a climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Pots were positioned in polypropylene boxes filled with water to the level of the substrate surface. Pot surfaces were covered with black, pinpricked polythene film (Aquafol, Reinmann, Emsdetten, Germany) to prevent growth of algae. Forty-five days after sowing, plants were harvested five hours after the beginning of the light period. Samples for expression profiling were harvested from the middle section of the blades of fully expanded green leaves and immediately frozen in liquid nitrogen.

extract_protocol:
PolyA+-RNA was extracted with magnetic beads (Dynabeads oligo (dT)25, Dynal, Oslo, Norway) following the manual instruction. In brief, 100mg leaf material was homogenized for 1.5 min at 28 Hz in a ball mill (Retsch) and 1.5 ml Lysis buffer (100 mM Tris, 500 mM LiCl, 10 mM EDTA, 1 % LiDS, 5 mM DTT, pH 8) was added. The solution was mixed and centrifugated at 4°C for 10min at 14000 rpm. 125µl beads were washed twice with 200µl Lysis buffer and were added to the supernatant. After gentle mixing for 5 min beads were washed twice with 1 ml washing buffer A (10 mM Tris, 150 mM LiCl, 1 mM EDTA, 0.1 % LiDS, 0.05% Tween, pH 8), beads were transferred to a new reaction vial and beads were washed twice with washing buffer B (10 mM Tris, 150 mM LiCl, 1 mM EDTA, 0.05% Tween, pH 8). PolyA+-RNA was extracted two times with 10 µl 10 mM Tris pH 7.5 heating 2 min to 95oC. For DNase treatment 20 µl RNA, 4 µl 5x Superscript III Puffer, 0.5 µl RNaseOUT (40 U, Invitrogen), 1 µl DNase (Roche) were incubated for 15 min at room temperature. After adding 1 µl 110 mM EDTA reaction was stopped by incubating for 10 min at 70°C.
mRNA from four plants originating from the same experiment, condition and cultivar was pooled. cDNA was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and purified by precipitation, using Bioline Sure Clean (Bioline, Luckenwalde, Germany).

treatment_protocol: see growth protocol

tag: Cultivar

value: IR57311-95-2-3 (IRGC accession 17509 (INGER)); Age:45 d; Tissue:middle section of the blades of fully expanded green leaves from 4 - 5 plants

label: Alexa Fluor  647

source_name: rice leaf;LC-93-4;drought

growth_protocol: Seeds were pre-germinated in tab water at 28°C for ten days. Then, plantlets were transferred in 10 cm diameter pots filled with 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany), an ion-exchange resin loaded with nutrient ions and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were transferred to a climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Pots were positioned in polypropylene boxes filled with water to the level of the substrate surface. Pot surfaces were covered with black, pinpricked polythene film (Aquafol, Reinmann, Emsdetten, Germany) to prevent growth of algae. Twenty-six days after sowing, water was removed from half of the boxes and plants were left to dry for four days, until the soil water content had reached the permanent wilting point (PWP) for 50% of the plants. Thereafter, the soil water content was kept constant to the fixed PWP value over a period of 14 days by weighing each pot at the end of the light period and adding the amount of water lost during the last 24 h. After a total of 19 days of drought stress, plants were harvested five hours after the beginning of the light period. Samples for expression profiling were harvested from the middle section of the blades of fully expanded green leaves and immediately frozen in liquid nitrogen.

value: LC-93-4 (LC); Age:45 d; Tissue:middle section of the blades of fully expanded green leaves from 4 - 5 plants


title: Rice moderate drought Rice22

description: no additional description

source_name: rice leaf;TaiPai;control

value: Taipai 309 (IRGC accession 42576); Age:45 d; Tissue:middle section of the blades of fully expanded green leaves from 4 - 5 plants


title: Rice moderate drought Rice25

description: no additional description

source_name: rice leaf;LC-93-4;control

source_name: rice leaf;IR 57311;drought


title: Rice moderate drought Rice27

description: no additional description

source_name: rice leaf;TaiPai;drought

source_name: rice leaf;Nipponbare;control

value: Nipponbare (IRGC accession 12731); Age:45 d; Tissue:middle section of the blades of fully expanded green leaves from 4 - 5 plants


title: Rice moderate drought Rice19

description: no additional description


title: Rice moderate drought Rice06

description: no further description

label_protocol: For labeling 1 mg cDNA (in 15 µl buffer (10 mM Tris, pH 8.5)) were mixed with 5 µl of the fluorescent dye (Alexa Fluor 532 and 647 (Qiagen, Hilden, Germany)) and incubated for 30 - 45 min at 85 °C in the dark, followed by adding of 5 µl Stop Solution and an incubation for 10 min at room temperature.)

growth_protocol: Seeds were pre-germinated in tab water at 28°C for ten days. Then, plantlets were transferred in 10 cm diameter pots filled with 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany), an ion-exchange resin loaded with nutrient ions and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were transferred to a climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Pots were positioned in polypropylene boxes filled with water to the level of the substrate surface. Pot surfaces were covered with black, pinpricked polythene film (Aquafol, Reinmann, Emsdetten, Germany) to prevent growth of algae. Twenty-six days after sowing, water was removed from half of the boxes and plants were left to dry for four days, until the soil water content had reached the permanent wilting point (PWP) for 50% of the plants. Thereafter, the soil water content was kept constant to the fixed PWP value over a period of 14 days by weighing each pot at the end of the light period and adding the amount of water lost during the last 24 h. After a total of 19 days of drought stress, plants were harvested five hours after the beginning of the light period. Samples for expression profiling were harvested from the middle section of the blades of fully expanded green leaves and immediately frozen in liquid nitrogen


title: Whole Larvae vs Larval Dorsal Vessel rep4

city: MARSEILLE Cedex 9

first: Bruno

last: ZEITOUNI

address: Campus de Luminy Case 907

institute: CNRS

description: Cy3 and Cy5 labelled aRNA samples were mixed in equal proportions (700 pmol) and fragmented with the RNA Fragmentation Reagents (Ambion) to enhance aRNA hybridization, dried up to 60ul of final volume, and hybridized on INDAC oligonucleotide microarrays.

hyb_protocol: Labeled probes (60 µl) were mixed with 60 µl of 4X hybridization buffer and 120 µl of deionised formamide, denatured at 95°C for 5min and hybridized at 37°C for 12 hours with the Automated Slide Processor (ASP) of Amersham. Slides were successively washed at 37°C in 1X SCC/0.2% SDS for 20 min, in 0.1X SCC/0.2% SDS for 2x10 min, in 0.1X SCC for 10 min, rinsed in isopropanol and air dried.

scan_protocol: Images were obtained with a GenePix 4200AL (Axon Instruments) at 10 um resolution and 5% of laser power. Fluorescence measurements are made using Array Vision quantification software (Imaging Research Inc).

data_processing:
Normalization of primary expression data (from not flagged spots) was performed through two successive steps using both R software packages SMA and LIMMA:
- Lowess normalization to normalize the M-values (Cy3/Cy5) for each array separately (within-array normalization) without prior background correction
- Quantile normalization to the A-values making the density distributions the same across arrays to compare expression intensities between them (between-array normalization).
Final normalized log2 M-values are showed in the VALUE column.

organism: Drosophila melanogaster

label_protocol:
20 μg of amino allyl aRNA was labelled according the "Dye Coupling and Labeled aRNA Cleanup" protocol of the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion)
700 pmol of incorporated dye was used for hybridization.

extract_protocol:
Total RNA was extracted from 5 whole third instar larvae with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Whole larvae were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy3.

tag: Strain

value: WT (yw; Canton S)

tag: Development temperature

tag: Stage

value: Third instar larvae

tag: Whole organism

tag: Biological replicate n°4

extract_protocol:
Total RNA was extracted from 5 dissected dorsal vessels with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Dissected entire cardiac tubes were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy5.

tag: Hand dissected dorsal vessel preparations


title: Larval Dorsal Vessel vs Whole Larvae rep2

extract_protocol:
Total RNA was extracted from 5 dissected dorsal vessels with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Dissected entire  cardiac tubes were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy3.

tag: Biological replicate n°2

extract_protocol:
Total RNA was extracted from 5 whole third instar larvae with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Whole larvae were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy5.


title: Rice moderate drought Rice10

description: no additional description

source_name: rice leaf;Nipponbare;drought


title: Rice moderate drought Rice09

description: no additional description


title: 13278975 - NonPol-Tr-1 vs NonPol-Co-1

hyb_protocol: NonPol-Co-1 Cy5 / NonPol-Tr-1 Cy3 : 30pmol. Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Rice moderate drought Rice23

description: no additional description


title: Rice moderate drought Rice05

description: no further description

growth_protocol:
Seeds were pre-germinated in tab water at 28°C for ten days. Then, plantlets were transferred in 10 cm diameter pots filled with 540 g sand mixed with 8 g of Lewatit HD 50 (Lanxess, Langenfeld, Germany), an ion-exchange resin loaded with nutrient ions and 0.4 g Fetrilon Combi (Compo, Münster, Germany). Pots were transferred to a climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Pots were positioned in polypropylene boxes filled with water to the level of the substrate surface. Pot surfaces were covered with black, pinpricked polythene film (Aquafol, Reinmann, Emsdetten, Germany) to prevent growth of algae. Twenty-six days after sowing, water was removed from half of the boxes and plants were left to dry for four days, until the soil water content had reached the permanent wilting point (PWP) for 50% of the plants. Thereafter, the soil water content was kept constant to the fixed PWP value over a period of 14 days by weighing each pot at the end of the light period and adding the amount of water lost during the last 24 h.
After a total of 19 days of drought stress, plants were harvested five hours after the beginning of the light period. Samples for expression profiling were harvested from the middle section of the blades of fully expanded green leaves and immediately frozen in liquid nitrogen.


title: GBS_DS101_CDM_rep3

city: Shreveport

first: Daniel

middle: Wilson

last: Shelver

state: LA

address: 1501 Kings Highway

department: Microbiology and Immunology

institute: LSU Health Sciences Center in Shreveport

description:
Biological replicate 3 of 3.
Scanned microarray images were analyzed using GeneChip Operating Software (GCOS) 1.4 (Affymetrix).

hyb_protocol: A hybridization cocktail containing 3.9 ug labelled cDNA was hybridized to the arrays. Hybridization was performed for 16 hours with a rotation of 60 rpm at a temperature of 45oC in a GeneChip Hybridization Oven 640 (Affymetrix). Arrays were washed and stained with a streptavidin-phycoerythrin conjugate using predefined protocols with a GeneChip Fluidics Station 450 (protocol ProkGE-WS2_450).

scan_protocol: Arrays were scanned with a GeneChip Scanner 300 7G (Affymetrix).

data_processing: Expression signals were calculated using the MAS5 statistical algorithm with the Affymetrix GCOS software version 1.4. Signal values were globally scaled to an intensity value of 500.

organism: Streptococcus agalactiae

source_name: Streptococcus agalactiae DS101, grown in chemically defined medium

label_protocol: cDNA was synthesized from 10 ug of total RNA using Superscript II reverse transcriptase (Invitrogen). Carryover RNA was degraded with 1 N NaOH followed by 1 N HCl. cDNA was purified and concentrated using a MinElute PCR Purification kit (Qiagen). Samples were fragmented with DNase I (Promega; Madison, WI) and biotin end-labelled with GeneChip DNA Labeling Reagent (Affymetrix).

growth_protocol: Group B Streptococcus (GBS) strains were cultured in chemically defined medium (CDM) with 400ug/ml Met to an O.D.600 = 0.3.

extract_protocol: 50 ml of cells (O.D.600 = 0.3) were harvested by centrifugation. GBS cells were then placed in a 950 ul suspension of QBio lysing matrix B, 50mM sodium acetate pH 4.0, 0.5% SDS, and 25% acidic phenol (Amresco; Solon, OH).  Cells were mechanically disrupted with the lysing matrix using a FastPrep® 120 device (MP Biomedicals; Solon, OH) with a speed setting of 5 and pulse length of 22 s.  The mixture was separated by centrifugation for 10 minutes.  RNA was precipitated by adding sodium acetate (pH 5.2) to a final concentration of 0.3M and adding 2 volumes of 100% ethanol.  One ml of Trizol reagent (Sigma-Aldrich) was added and incubated at room temperature for 5 min.  200 ul of chloroform was added, shaken vigorously, and allowed to remain at room temperature for 2 minutes. The mixture was centrifuged for ten minutes, the aqueous phase was recovered and RNA was precipitated with an equal volume of 100% isopropanol.  The pellet was air-dried and resuspended in 50 ul DEPC-treated water.  Residual DNA was removed using DNaseI (Promega) and subsequent clean-up with a RNEasy mini spin column (Qiagen).

tag: Strain DS101, derivative of wild-type strain COH1 harboring mtaR::Kan mutation.


title: GBS_DS101_CDM_rep2

description:
Biological replicate 2 of 3.
Scanned microarray images were analyzed using GeneChip Operating Software (GCOS) 1.4 (Affymetrix).


title: GBS_COH1_CDM_rep1

description:
Biological replicate 1 of 3.
Scanned microarray images were analyzed using GeneChip Operating Software (GCOS) 1.4 (Affymetrix).  The absolute call (present, marginal, absent) of each gene expression in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software.

hyb_protocol: A hybridization cocktail containing 3.9 ug labelled cDNA was hybridized to the arrays.  Hybridization was performed for 16 hours with a rotation of 60 rpm at a temperature of 45oC in a GeneChip Hybridization Oven 640 (Affymetrix).  Arrays were washed and stained with a streptavidin-phycoerythrin conjugate using predefined protocols with a GeneChip Fluidics Station 450 (protocol ProkGE-WS2_450).

source_name: Streptococcus agalactiae COH1, grown in chemically defined medium

label_protocol: cDNA was synthesized from 10 ug of total RNA using Superscript II reverse transcriptase (Invitrogen).  Carryover RNA was degraded with 1 N NaOH followed by 1 N HCl.  cDNA was purified and concentrated using a MinElute PCR Purification kit (Qiagen).  Samples were fragmented with DNase I (Promega; Madison, WI) and biotin end-labelled with GeneChip DNA Labeling Reagent (Affymetrix)

tag: Strain COH1, wild-type clinical isolate.


title: Larval Heart vs Larval Aorta rep4

extract_protocol:
Dorsal vessels were hand dissected from third larval instar individuals, and aorta and heart were separated at their boundary, the cardiovascular valve.
Total RNA was extracted from 10 dissected hearts with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Dissected hearts were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy3.

tag: Hand dissected heart preparations

extract_protocol:
Dorsal vessels were hand dissected from third larval instar individuals, and aorta and heart were separated at their boundary, the cardiovascular valve.
Total RNA was extracted from 10 dissected aortae with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Dissected aortae were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy5.

tag: Hand dissected aorta preparations


title: GBS_COH1_CDM_rep2

data_processing: Expression signals were calculated using the MAS5 statistical algorithm with the Affymetrix GCOS software version 1.4.  Signal values were globally scaled to an intensity value of 500.


title: GBS_DS101_CDM_rep1

description:
Biological replicate 1 of 3.
Scanned microarray images were analyzed using GeneChip Operating Software (GCOS) 1.4 (Affymetrix).

label_protocol: cDNA was synthesized from 10 ug of total RNA using Superscript II reverse transcriptase (Invitrogen).  Carryover RNA was degraded with 1 N NaOH followed by 1 N HCl.  cDNA was purified and concentrated using a MinElute PCR Purification kit (Qiagen).  Samples were fragmented with DNase I (Promega; Madison, WI) and biotin end-labelled with GeneChip DNA Labeling Reagent (Affymetrix).


title: Larval Dorsal Vessel vs Whole Larvae rep1

tag: Biological replicate n°1


title: Rice moderate drought Rice21

description: no additional description


title: Larval Heart vs Larval Aorta rep3

tag: Biological replicate n°3


title: Rice moderate drought Rice20

description: no additional description


title: Larval Aorta vs Larval Heart rep2

extract_protocol:
Dorsal vessels were hand dissected from third larval instar individuals, and aorta and heart were separated at their boundary, the cardiovascular valve.
Total RNA was extracted from 10 dissected aortae with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Dissected aortae were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy3.

extract_protocol:
Dorsal vessels were hand dissected from third larval instar individuals, and aorta and heart were separated at their boundary, the cardiovascular valve.
Total RNA was extracted from 10 dissected hearts with Trizol reagent (Invitrogen) in following a modified manufacturer’s experimental protocol adapted to the extraction of small RNA amounts. Dissected hearts were collected in 300 µl of Trizol and a linear polyacrylamide (GenElute LPA from Sigma) was used as nucleic acid carrier (necessary for small amounts of starting material).
Isolated total RNA (~100 ng) was directly amplified with the Amino Allyl MessageAmpTM II aRNA Amplification Kit (Ambion): the aRNA procedure begins with total RNA that is reverse transcribed using an oligo(dT) primer containing a T7 RNA polymerase promoter sequence. The reaction is treated with RNase H to cleave the mRNA into small fragments. These small RNA fragments serve as primers during the second-strand synthesis reaction producing a double-stranded cDNA template for T7 in vitro transcription (IVT). This RNA was subjected to a second round of amplification with a second IVT configured to incorporate the modified nucleotide (amino allyl UTP) into the aRNA during transcription for subsequent indirect labelling with the fluorescent dye Cy5.


title: Rice moderate drought Rice04

description: no further description


title: Whole Larvae vs Larval Dorsal Vessel rep3


title: Snf1-I132G+2NMPP1_Raffinose_1

status: Public on Oct 27 2008

city: Pittsburgh

first: Karen

middle: M.

last: Arndt

state: PA

address: 4249 Fifth Avenue

department: Biological Sciences

institute: University of Pittsburgh

scan_protocol: Affymetrix GeneChip Fluidics Station 450 and DAT files generated by Microarray Analysis Suite 5.0 (MAS5)

data_processing: MAS 5.0; scaled to target of 500; default setting used.

label: Biotin

label_protocol: per Manufacturer's instructions-BioArray High Yield RNA Transcript Labeling Kit (Enzo)

growth_protocol: grown in SC-ura medium+2% glucose to early log phase and then transferred to 2% raffinose + 0.05% glucose for 120 min. before adding 25 micromolar 2NM-PP1 for 40 min

extract_protocol: Total RNA prepared by glass bead lysis, digested with DNase and purified by RNeasy Kit (Qiagen). Sample quality assessed by BioAnalyzer (Agilent).

treatment_protocol: 25 micromolar 2NM-PP1 for 40 min

tag: PY133 (Mat a his3del200 ura3-52 snf1del) transformed with HA-SNF1-I132G allele


title: Larval Aorta vs Larval Heart rep1


title: Snf1+2NMPP1_Raffinose_2

hyb_protocol: Affymetrix GeneChip Fluidics Station 450

tag: PY133 (Mat a his3del200 ura3-52 snf1del) transformed with HA-SNF1 allele


title: Snf1-I132G+2NMPP1_Raffinose_2


title: Snf1+2NMPP1_Raffinose_1