title: wnt4_wt1

description:
>> Amplification protocol <<
Target Amplified manufacturer/kit: Affymetrix (Enzo)
Target Amplified protocol: Affymetrix Standard
Rounds of amplification:  1
URL: http://gudmap.hgu.mrc.ac.uk/gudmap/pages/mic_submission.html?id=GUDMAP:7050

tag: developmental stage

tag: theiler stage


title: XX_WT-P_081407_A

status: Public on Mar 01 2009

type: RNA

city: Berkeley

first: Barbara

middle: J.

last: Meyer

state: CA

address: 16 Barker Hall #3204

department: MCB

country: USA

institute: HHMI/UCB

hyb_protocol: Following fragmentation, 15 microg of cRNA were hybridized for 16 hr at 45C on GeneChip C. ElegansGenome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix Scanner 30007G.

data_processing: Arrays were normalized by RMA (Robust Multichip Average) using Affymetrix Expression Consol software (Irizarry et al. 2003).    Data were analyzed by ArrayStar.  Statistical analysis was performed using a moderated t-test with FDR multiple testing correction (Benjamini-Hochberg method). Irizarry, R.A., Hobbs, B., Collin, F., Beazer-Barclay, Y.D., Antonellis, K.J., Scherf, U., and Speed, T.P. 2003. Exploration, Normalization, and Summaries of High Density Oligonucleotide Array Probe Level Data. Biostatistics 4: 249-264.

label: biotin

molecule: total RNA

organism: Caenorhabditis elegans

source_name: embryo from worms grown on RNAi plates

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 8 microg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: sdc-2(-) XX animals were prepared by growing sdc-2(y93) XX animals on Ahringer feeding library bacteria (Kamath et al. 2003) bearing an sdc-2 plasmid.  The wild-type control for the sdc-2 RNAi was N2 worms fed Ahringer library feeding bacteria bearing a plasmid lacking an insert.  Feeding bacteria was prepared by seeding 1L LB cultures with a single colony, growing overnight at 37∞C, inducing for 2hr with 5 mM IPTG, pelleting, and resuspending in 1 volume (w/v) of LB with 20% glycerol.

extract_protocol: Gravid adults were bleached and embryos harvested as described (Portman, D.S, Wombook, Profiling C. elegans gene expression with DNA microarrays) and 100 µl aliquots snap frozen in liquid nitrogen and stored at -80∞C.  An aliquot was thawed at 65∞C for 10 minutes, resuspended in 1ml Trizol (Invitrogen, Carlsbad, CA), vortexed vigorously to resuspend, and incubated for 20 minutes at room temperature. The sample was then centrifuged for 15 minutes at 12,000xg at 4∞C.  The aqueous phase was transferred to a new tube and 100 µl of BCP (1-bromo-3-chloropropane) (MRC, Cincinnati, OH) was added, the sample was mixed by vortexing, incubated for 15 minutes at room temperature, and centrifuged for 10 minutes at 12,000xg at 4∞C.  The aqueous phase was transferred to a new tube and nucleic acid was precipitated by addition of 500 µl of isopropanol. The sample was incubated for 10 minutes at room temperature, centrifuged at 12,000xg for 8 minutes, washed in 70% ethanol and air dried for 5 minutes.  Nucleic acid was resuspended in 100 µl nuclease-free water and RNA quantified with a UV spectrophotometer. One hundred micrograms of RNA was then treated with 3 µl RQ1-DNAse (Promega, Madison, WI) and RNA isolated with the RNAeasy kit as per the manufacturer's protocol (Qiagen, Valencia, CA). RNA was again quantified with a UV spectrophotometer and cDNA was prepared from 5 µg RNA using the Protoscript Kit (NEB, Ipswich, MA) and diluted 1:15 in water.

treatment_protocol: Embryos used for microarray analysis were obtained from bleached gravid adults.

tag: Wild Type controls for sdc-2


title: wnt4_wt2

status: Public on Nov 18 2006

city: Edinburgh

first: GUDMAP

last: Developers

address: Crewe Road

country: United Kingdom

institute: IGMM MRC Human Genetics Unit

description:
>> Amplification protocol <<
Target Amplified manufacturer/kit: Affymetrix (Enzo)
Target Amplified protocol: Affymetrix Standard
Rounds of amplification: 1
URL: http://gudmap.hgu.mrc.ac.uk/gudmap/pages/mic_submission.html?id=GUDMAP:7051

hyb_protocol: Amount labeled  target hybridization to array: 15 ug Hybridization/Wash/Stain protocol: EukGE-WS2v4

scan_protocol: Affymetrix standard protocol

data_processing: Analysis method: Affymetrix GCOS and Rosetta Resolver GCOS Tgt value: 500

label: Biotin

organism: Mus musculus

source_name: Metanephros

label_protocol: Affymetrix standard protocol

extract_protocol: Disruption in liguid nitrogen, total RNA isolation with Qiagen Rneasy, followed by homogenization with QiaShredder columns (two 30ul elutions).

treatment_protocol:
pool size: 2 wild-type kidneys
Pooled sample: Yes
Dissection Method: Whole organ excision
Experimental Design: Wnt4 is required for renal vesicle (RV) induction. Therefore, RV and the derivatives (s-shaped body and eventually the mature nephron) are missing in Wnt4 mutants. At E14.5, these structures are present in wildtype kidneys. Transcriptional profile comparison between E14.5 wildtype and Wnt4 mutants therefore identify genes expressed in the RV and derivatives. Minimally pooled kidney samples were used as single biological replicates. CD-1 mice are mated overnight and examined the following morning for the presence of a copulatory plug. Presence of a plug is taken as day 0.5 post coitum. Pregnant CD-1 mice are euthanized by standard carbon dioxide asphyxiation. All fetuses are euthanized by decapitation with scissors. Fetal kidneys are removed from stage E14.5 embryos and stored in RNAlater at 4°C until RNA isolation (<4 days). Total RNA was isolated from these pools and subjected to a single round of amplification for use on Affymetrix arrays.

tag: gene reported

value: wild-type (PMID:18346943)

tag: strain

tag: sex

value: unknown

tag: somite count

tag: developmental landmark


title: collecting duct - xur3

description:
>> Amplification protocol <<
Target Amplified manufacturer/kit: Affymetrix (Enzo)/Arcturus
Target Amplified protocol: Arcturus/Affymetrix
Rounds of amplification: 2
URL: http://gudmap.hgu.mrc.ac.uk/gudmap/pages/mic_submission.html?id=GUDMAP:7064

source_name: collecting duct

extract_protocol: Total RNA extracted using TRIzol.  Two round amplification using the Arcturus kit according to the manufacturer protocol.

treatment_protocol:
pool size: 1 collecting duct
Pooled sample: No
Dissection Method: Whole organ excision
Two early nephron structures, the renal vesicle and the s-shaped body, and the collecting duct system, were microdissected from E14.5 kidneys based on morphological criteria.  Antibody and lectin staining together with RT-PCR were used for identity verification and for exclusion of contamination by other tissues.  Samples are minimally pooled for amplification.  A biological replicate is a single minimally pooled sample.  RNA isolated from the collected structures was put through two rounds of T7-driven amplification to obtain the microgram quantities of cRNA needed for hybridization to Affymetrix microarrays.

value: wild-type

tag: Sex


title: wnt4_wt4

description:
>> Amplification protocol <<
Target Amplified manufacturer/kit: Affymetrix (Enzo)
Target Amplified protocol: Affymetrix Standard
Rounds of amplification: 1
URL: http://gudmap.hgu.mrc.ac.uk/gudmap/pages/mic_submission.html?id=GUDMAP:7053


title: SKI-DLCL_tumor_animals treated with 1mg/kg Aplidin_50mg/kg AraC

status: Public on Sep 15 2006

city: New Brunswick

first: Pravin

last: Mishra

state: NJ

address: 195 Little Albany St.

department: Pharmacology

institute: UMDNJ

description: Sample processed by CINJ core expression array facility according to Affymetrix protocol.

hyb_protocol: Hybridization  Water, Molecular Biology Grade or DEPC-treated Acetylated Bovine Serum Albumin (BSA) solution (50 mg/mL) Herring Sperm DNA GeneChip Eukaryotic Hybridization Control Kit 5M NaCl, RNase-free, DNase-free MES Free Acid Monohydrate MES Sodium Salt EDTA Disodium Salt  Miscellaneous Reagents/Supplies  Surfact-Amps 20 (Tween-20), 10% Hybridization Oven 640 Sterile, RNase-free, microcentrifuge tubes, 1.5mL Micropipettors Sterile-barrier pipette tips and non-barrier tips Heatblock

scan_protocol: Washing, Staining, and Scanning  Water, Molecular Biology Grade Distilled water Acetylated Bovine Serum Albumin (BSA) solution (50 mg/mL) R-Phycoerythrin Streptavidin 20X SSPE (3M NaCl, 0.2M NaH2PO4, 0.02M EDTA) Goat IgG, Reagent Grade Anti-streptavidin antibody (goat), biotinylated 10% surfact-Amps 20 (Tween-20)

data_processing: scanned fluorescence intensity values, not normalized

organism: Homo sapiens

source_name: RNA isolated from SKI-DLCL tumor harvested from mice treated with Aplidin and AraC

label_protocol:
Synthesis of Biotin-Labeled cRNA/Purification

ENZO BioArray RNA Transcript Labeling Kit
GeneChip Sample Cleanup Module

Miscellaneous Reagents/Supplies

100% ethanol room temperature
80%   ethanol room temperature
Water, Molecular Biology Grade or DEPC-treated
Ethidium Bromide
Sterile, RNase-free, microcentrifuge tubes, 1.5mL
Micropipettors
Sterile-barrier, RNase-free pipette tips
Mini agarose gel electrophoresis unit with appropriate buffers
UV Spectrophotometer
Cooling water bath

growth_protocol: Mice were kept in CINJ vivarium under standard condition. SKI-DLCL cells were injected s.c.

extract_protocol: RNeasy kit from Qiagen

treatment_protocol: Mice were given a single dose of 1mg/kg Aplidin and 50mg/kg AraC.

tag: SKI-DLCL tumor grown in CB-17 scid/scid mice. Tumor was harvested on day eight following the treatment. Total RNA was extracted using RNeasy kit.


title: SKI-DLCL cells_Aplidin treated 3nM_24h_rpt3

description: Samples were processed in CINJ Core Expression Array Facility according to Affymetrix protocol.

data_processing: scanned fluorescence intensity, no normalization

source_name: SKI-DLCL cells

label_protocol:
Synthesis of Biotin-Labeled cRNA/Purification

ENZO BioArray RNA Transcript Labeling Kit
GeneChip Sample Cleanup Module

Miscellaneous Reagents/Supplies

100% ethanol ? room temperature
80%   ethanol ? room temperature
Water, Molecular Biology Grade or DEPC-treated
Ethidium Bromide
Sterile, RNase-free, microcentrifuge tubes, 1.5mL
Micropipettors
Sterile-barrier, RNase-free pipette tips
Mini agarose gel electrophoresis unit with appropriate buffers
UV Spectrophotometer
Cooling water bath

growth_protocol: Cells were grown under standard cell culture condition (95% air, 5% CO2, 37C)

extract_protocol: Total RNA was extracted using RNeasy kit from Qiagen.

treatment_protocol: Aplidin alone at concentration 3 nM

tag: SKI-DLCL cells were treated with Aplidin at concentration 3nM for 24h. Total RNA was extracted.


title: SKI-DLCL_tumor_untreated animals

description: Protocol taken from the web site of CINJ Core Expression Array Facility where the samples were processed.

hyb_protocol: Hybridization -Thaw components of the Eukaryotic Hybridization Control Kit. Heat 20X Eukaryotic Hybridization Control to 65°C for 5 minutes to denature cRNA controls. Equilibrate desired probe array to room temperature. In a new, sterile RNase-free 1.5mL microfuge tube, add the following components:  -20µl fragmented cRNA -5µl Control Oligonucleotide B2 (3nM) -15µl 20X Eukaryotic Hybridization Controls -3µl Herring Sperm DNA (10mg/mL) -3µl Acetylated BSA (50mg/mL) -150µl 2X Hybridization Buffer -104µl RNase-free water  Total hybridization volume is 300µl  Heat hybridization cocktail to 95°C for 5 minutes, followed by heating at 45°C for 5 minutes. In a new, sterile RNase-free 1.5mL microfuge tube, combine a volume of 2X Hybridization Buffer with an equal volume of RNase-free water (make 1X Hybridization Buffer). Wet the probe array by filling it with 250µl of 1X Hybridization Buffer and incubate in Hybridization Oven 640 set at 45°C for 10 minutes with rotation setting at 60rpm. Centrifuge hybridization cocktail for 5 minutes at max. speed to spin down any insoluble material. Remove 1X Hybridization Buffer from probe array and fill with 250µl of hybridization cocktail, taking care not to pipette any insoluble material. Place probe array in Hybridization Oven 640, heated to 45°C and with rotation setting at 60rpm for 16 hours.

scan_protocol: Washing, Staining, and Scanning  Stain  For each array, in a new, sterile RNase-free 1.5mL microfuge tube, combine the following:  600.0µl of 2X MES Stain Buffer 48.0µl Acetylated BSA (50mg/mL) 12.0µl Streptavidin Phycoerythrin (SAPE) (1mg/mL) 540.0µl water, Molecular Biology Grade  Total volume is 1200µl.  Divide into two aliquots of 600µl each to be used for stains 1 and 3.  Antibody For each array, in a new, sterile RNase-free 1.5mL microfuge tube, combine the following:  300.0µl 2X MES Stain Buffer 24.0µl Acetylated BSA (50mg/mL) 6.0µl Norma Goat IgG (10mg/mL) 3.6µl Biotinylated antibody (0.5mg/mL) 266.4µl water, Molecular Biology Grade  Total volume is 600µl.  After 16 hours of hybridization, remove 250µl of sample (in hybridization cocktail) and put back into eppendorf, containing fragmented cRNA sample. Fill the array with 250µl of Non-Stringent Wash Buffer (Wash A)  Now ready for Fluidics Station, followed by scanning.

data_processing: scaned fluorescence intensity values, no normalization

source_name: SKI-DLCL tumor in CB-17 scid/scid mice, untreated, control

label_protocol:
Synthesis of Biotin-Labeled cRNA
Thaw components of the ENZO BioArray RNA Transcript Labeling Kit and purified cDNA mixture from the previous day.
In a new, sterile, 1.5mL RNase-free microfuge tube, add the following components:

10µl purified cDNA
12µl RNase-free water
4µl 10X HY Reaction Buffer (vial#1)
4µl 10X Biotin-Labeled Ribonucleotides (vial#2)
4µl 10X DTT (vial#3)
4µl 10X RNase Inhibitor Mix (vial#4)
2µl 20X T7 RNA Polymerase (vial#5)

Total reaction volume is 40µl

Gently mix and spin down.
Incubate in a 37°C water bath for 4 hours.

Cleanup of IVT Reaction

Take out 1ul of the reaction mix for gel electrophoresis inspection.
Using the GeneChip Sample Cleanup Module, add 60µl of RNase-free water to the reaction.
Mix for 3 seconds.
Add 350µl IVT cRNA Binding Buffer and mix for 3 seconds.
Add 250µl of 100% ethanol and mix well (do not centrifuge).
Add sample (~700ul total) to the IVT cRNA Cleanup Spin Column sitting in a 2mL Collection Tube.
Centrifuge for 15 seconds at 10,000rpm.
Discard flow-through and Collection Tube.
Transfer spin column to a new 2mL Collection Tube.
Add 500µl IVT cRNA Wash Buffer to spin column.
Centrifuge for 15 seconds at 10,000rpm.
Discard flow-through.
Add 500µl of 80% ethanol onto spin column.
Centrifuge for 15 seconds at 10,000rpm.
Discard flow-through.
Open the cap of the spin column and centrifuge for 5 minutes at max. speed.
Discard flow-through and Collection Tube.
Transfer spin column to a new 1.5mL Collection Tube and pipet 11µl of RNase-free water directly onto the spin column membrane.
Centrifuge for 1 minute at max. speed to elute.
Add another 10µl of RNase-free water directly onto spin column membrane and centrifuge for 1 minute at max. speed to elute.

Use spectrophotometric analysis to determine cRNA yield and purity using a 1:100 dilution of purified cRNA (A260/A280 ratio should be close to 2.0 for pure RNA).

Calculate the adjusted cRNA yield using the following:

adjusted cRNA yield = RNAm (total RNAi)(y)

RNAm = amount of cRNA measured after IVT (µg)
total RNAi = starting amount of total RNA (µg)
y = fraction of cDNA reaction used in IVT

For fragmentation, adjusted cRNA concentration must be at least 0.6µg/µl.
The final concentration of RNA in the fragmentation mix can range from 0.5µg/µl to 2µg/µl.

For standard arrays, 15µg of fragmented cRNA is required.
Fragmentation
In a thermal cycler compatible microfuge tube, combine 4µl 5X Fragmentation Buffer (supplied in GeneChip Sample Cleanup Module), 15µg cRNA (variable) and RNase-free water for a final volume of 20µl.
Incubate in a thermal cycler at 94°C for 35 minutes.
Place mixture on ice.
Take out 1ul of fragmented cRNA for gel electrophoresis inspection.

Run crude cRNA and fragmented cRNA samples, along with a 1Kb ladder on a 1% agarose gel for visual inspection.

growth_protocol: mice were grown in CINJ facility, standard condition

treatment_protocol: not treated

tag: Tumor was harvested from animals at day eight of the treatment. RNA was extracted using RNeasy kit.


title: LD2a

status: Public on Jul 29 2004

city: College Station

first: Phillip

middle: D.

last: Beremand

state: TX

department: Biology

institute: Texas A&M University

description: The LD2a sample is one of 4 identically obtained samples from 4 different sets of chickens.

hyb_protocol: Hybridizations and washes were as suggested by Genisphere.

scan_protocol: Arrays were scanned in an Affymetrix 428 array scanner.

data_processing: The images obtained were subsequently analyzed by GenePixPro software (Axon Instruments). The GenePix results (.gpr) file was further analyzed by GeneSpring 5 (Silicon Genetics) which applied intensity dependent LOWESS normalization to the results and calculated normalized Cy5 to Cy3 ratios.

organism: Gallus gallus

source_name: Chicken retina collected at LD 2

label_protocol: Randomly primed fluorescent probes were produced from the aRNA samples using a Genisphere 3DNA Array 900MPX expression array detection kit. The fluorescent dye on the probe derived from the experimental aRNA (LD2) was Cy5 while the dye on the control probe (LD18) was Cy3.

extract_protocol:
Cy5
Total RNA was extracted from each set of retinas using Trizol Reagent (Invitrogen). The total RNA samples were amplified to produce aRNA using the Message Amp Kit (Ambion).

source_name: Chicken retina collected at LD 18

extract_protocol:
Cy3
Total RNA was extracted from each set of retinas using Trizol Reagent (Invitrogen). The total RNA samples were amplified to produce aRNA using the Message Amp Kit (Ambion).


title: 181C3

status: Public on Jul 04 2005

city: Galveston

first: Allan

last: Brasier

address: 301 Univ. Blvd

department: Medicine

institute: University Texas Medical Branch

description: Cells expressing IkBa dominant negative cultured in absence of dox, TNF stimulation for 3 h

source_name: HeLa cell


title: 181C1

description: Cells expressing IkBa dominant negative cultured in absence of dox, TNF stimulation for 1 h


title: LD2b

status: Public on Sep 11 2004

description: The LD2b sample is one of 4 identically obtained samples from 4 different sets of chickens.

source_name: Chicken retinas from LD2

source_name: Chicken retinas from LD18


title: 180C6

description: Cells expressing IkBa dominant negative cultured in absence of dox, TNF stimulation for 6 h


title: LD2c

status: Public on Sep 16 2004

description: The LD2c sample is one of 4 identically obtained samples from 4 different sets of chickens.

source_name: chicken retinas from LD18


title: A. thal leaf tissue

type: SAGE

anchor: NlaIII

city: Saskatoon

first: Dustin

last: Cram

country: Canada

institute: Agriculture and Agri-Food Canada - Saskatoon Research Centre

description: Arabidopsis thaliana (Col-4) aerial tissue collected from 14-day old plants.  Grown under sterile conditions at 22 degrees Celsius and 125 uE light on 0.5xMS media.

organism: Arabidopsis thaliana

source_name: aerial tissue


title: Leaf Transcriptome of Zea mays Using RL-SAGE Method

status: Public on Sep 23 2004

city: Columbus

first: Guo-Liang

last: Wang

state: OH

address: 2021 Coffey Road

department: Plant Pathology

institute: Ohio State University

description:
Using the RL-SAGE method (Gowda et al. 2004), a maize leaf longSAGE library (cv. inbred line B73) was constructed. Leaf tissues were harvested from 4-week old B73 plants for RNA isolation. The conditions in the growth chamber were 12 h light (500 µmol photons m-2 sec-1), 20oC at night, 26oC in the day and 85% relative humidity. A total of 44,870 unique tags (17 bases +CATG) were identified from 232,948 individual tags in the maize leaf library.
Gowda M, Jantasuriyarat C, Dean RA, Wang GL (2004) Robust-LongSAGE (RL-SAGE): A Substantially Improved LongSAGE Method for Gene Discovery and Transcriptome Analysis. Plant Physiol 134(3):890-7
Lot batch =

organism: Zea mays

source_name: 30-day-old leaves