"Investigation Title" "FDA-CDER MTS RNA reagent cross platform test" "Experimental Design" "quality_control_testing_design" "Experimental Factor Name" "PLATFORM" "INDIVIDUAL" "MIX" "OPERATOR" "Experimental Factor Type" "platform" "individual" "mix" "operator" "Person Last Name" "Pine" "Person First Name" "Scott" "Person Mid Initial" "Person Affiliation" "FDA-CDER" "Person Roles" "submitter" "Public Release Date" "2005-11-23" "Comment[ArrayExpressSubmissionDate]" "2005-06-01" "Publication Author List" "Publication Title" "Experiment Description" "This experiment is a test of a reagent that can provide a basis for performance assessments on Affymetrix RAE230A, CodeLink UniSet Rat I, and Agilent G4130A rat oligonucleotide arrays. The reagent is a set of two mixed tissue RNA samples formulated to contain different ratios of RNA for each of four rat tissues (brain, liver, kidney, and testes). The analytes in this assay bind to a subset of gene probes that were matched across platforms to the same UniGene cluster or GenBank accession number, were determined to be tissue-selective on each of the 3 platforms using average baseline expression levels in single tissue samples from control animals, and have signal intensities that span the dynamic range of each platform. This reagent forms the basis of a ratiometric assay in a complex biological background that can be used to objectively assess the effect of different array processing methods and conditions on microarray data comparability within and across platforms, and could be a paradigm for similar performance standards for mouse and human arrays." "Protocol Name" 6377 6380 "6396-mix1" "6396-mix2" 6407 6409 6486 6489 6491 6552 6553 6672 6673 "6841-mix1" "6841-mix2" 7671 7672 8188 8189 8190 8309 8532 8534 8535 8536 8537 8538 8573 8588 8589 8590 "Protocol Type" "grow" "nucleic_acid_extraction" "pool" "pool" "image_aquisition" "image_aquisition" "labeling" "labeling" "hybridization" "bioassay_data_transformation" "bioassay_data_transformation" "nucleic_acid_extraction" "grow" "pool" "pool" "labeling" "labeling" "hybridization" "hybridization" "image_aquisition" "image_aquisition" "labeling" "labeling" "labeling" "labeling" "labeling" "labeling" "hybridization" "hybridization" "hybridization" "image_aquisition" "Protocol Description" "Rodents received certified rodent diet #5002C ad lib and drinking water purified by reverse osmosis. The animals were acclimated for 6 days prior to euthanasia. The animals were on a 12 hr light/dark cycle and euthanasia was performed consistently within 4-6 hr after the start of the light cycle. Each batch was made up of 8 animals from the same shipment. " "After euthanasia in a slow charged CO2 chamber, the rats were immediately decapitated to allow for rapid access to the brain. Four organs were quickly removed in the following order: brain, liver, kidneys, and testes. The whole brain, including brain stem but excluding pituitary gland, was collected. Tissues were quickly dissected into 0.5 cm sections while submersed in RNAlater (Ambion, Austin, TX) in sterile petri plates and placed into 50 mL tubes containing RNAlater at a ratio of 10 mL of RNAlater per 1g of tissue. Tissues were stored at 4�°C for a minimum of 24 hrs and a maximum of 72 hrs.

All tissue RNAs were isolated using a Tempest rotor-stator homogenizer (VirTis, Gardiner, NY) and Qiagen RNA isolation kits (Qiagen, Chatsworth, CA), following the manufacturer�s protocol. Brain RNA was isolated using QIAzol reagent, followed by a cleanup step with an RNeasy Maxi kit. Kidney, liver, and testes were homogenized in 15 ml RLT buffer per mg of tissue, diluted to 30 ml RLT per mg, and isolated on RNeasy Maxi columns using 15 ml homogenate per column. After an additional cleanup step on RNeasy Maxi columns, RNA was aliquoted and stored at -70�°C.

" "Mix1 consisted of 200 ug testis RNA, 600 ug liver RNA, 800 ug brain RNA, and 400 ug kidney RNA. 50 ug aliquots of each mixture were frozen at -70C." "Mix2 consisted of 800 ug testis RNA, 400 ug liver RNA, 400 ug brain RNA, and 400 ug kidney RNA. 50 ug aliquots of each mixture were frozen at -70C." "GeneChips were scanned on a GeneArray 2500 scanner using MicroArray Suite v. 5.0.0.032 and default scanner settings." "GeneChips were scanned on a GeneChip 3000 scanner using default scanner settings." "1st strand cDNA synthesis:

For each sample, add

2 ul T7-(dT)24 primer @ 50 uM (Affymetrix PN 900375) to

5 ug (up to 8 ul) total RNA.

Incubate @ 70C, 10 minutes, quick spin, quench on ice.

Make the following cocktail for 5 reactions:

20 ul 5X 1st Strand cDNA Buffer (Invitrogen PN 18064014),

10 ul 0.1 M DTT, and

5 ul 10 mM dNTP mix (Invitrogen PN 18427013).

Add 7 ul cocktail to each reaction. Incubate @ 42C for 2 minutes. Check volume.

To each tube, add 1 ul SuperScript II RT (200 U/ul) (Invitrogen PN 18064014).

Mix by pipetting , incubate @ 42 C for 1 hour in a PCR machine.

Total volume should now be 20 ul.

Second strand cDNA synthesis:

Place 1st strand reactions on ice. Spin briefly in microfuge.

Make the following cocktail for 5 reactions and add 130 ul of the cocktail to each sample:

455 ul DEPC-H2O, 150 ul 5X Second Strand Reaction Buffer (Invitrogen PN 10812014), 15 ul 10 mM dNTP Mix (Invitrogen PN 18427013), 5 ul E.Coli DNA Ligase (10 U/ul) (Invitrogen PN 18052019), 20 ul E.Coli DNA Polymerase I (10 U/ul) (Invitrogen PN 18010025),and 5 ul E.Coli RNase H (2 U/ul) (Invitrogen PN 18021071) for 650 ul total Volume

Gently tap tube to mix. Spin Briefly. Incubate @ 16C for 2 hrs in a PCR machine.

Add 2 ul T4 DNA Polymerase (10 U) (Invitrogen PN 18005025). Mix by pipetting. Incubate @ 16C for 5 minutes in a PCR machine.

Add 10 ul 0.5 M EDTA (Invitrogen PN 15575020) to a fresh 1.5 ml Eppendorf tube. Then add the above reaction to this tube to stop the reaction. Proceed to clean up OR store @ -20C for later use.

Cleanup of Double-Stranded cDNA using Sample Cleanup Module (Affymetrix PN 900371):

Transfer entire sample (162 ul) to a 1.5 mL sterile microfuge tube. Add 600 ul cDNA Binding Buffer.

Check that the color of the mixture is yellow, if not, add 10 ul of 3M sodium acetate, pH 5.0, and mix.

Apply 500 ul of sample to cDNA Cleanup spin Column, and spin @ 10,000 x g, 1 minute. Discard flow-through.

Reload remainder of sample (262 ul), and spin again, discard flow-through. Transfer spin column to a new 2 mL Collection Tube. Pipet 750 ul cDNA Wash Buffer onto the spin column. Spin @ 10,000 x g, 1 minute. Discard flow-through.

Open the cap of the spin column and spin 5 minutes at 18,000 x g to dry the column. Discard flow-through and Collection Tube.

Transfer spin column to a fresh 1.5 mL Collection Tube, and pipet 14 uL of cDNA Elution Buffer directly to membrane. Incubate for 1 minute @ RT. Spin 1 minte 18,000 x g to elute.

Synthesis of Biotin-Labled cRNA using Enzo Bioarray HighYield RNA Transcript Labeling Kit (Affymetrix PN 900182):

Add 10 ul Template ds-cDNA to a new 0.5 mL Rnase-free microfuge tube. Prepare cocktail by adding the following reagents in order, then add 30 ul of cocktail to 10 ul of ds-cDNA sample:

60 ul DEPC-H2O, 20 ul 10X HYB Reaction Buffer (Vial 1), 20 ul 10X Biotin Labeled Ribonucleotides (Vial 2), 20 ul 10X DTT (Vial 3), 20 ul 10X Rnase Inhibitor Mix (Vial 4), and 10 ul T7 RNA Polymerase (Vial 5) for 150 ul total.

Carefully mix the reagents and spin briefly. Incubate @ 37C for 4-5 hrs in a PCR machine, gently mix the contents every 45 minutes during incubation.

Proceed to Cleanup OR store @ -20C for future use.

Cleanup and Quantification of Biotin-Labled cRNA:

Transfer sample to a 1.5 mL RNase-free microfuge tube. Add 60 ul of DEPC-H2O to the in vitro transciption reaction and mix by vortexing 3 seconds.

Add 350 ul IVT cRNA Binding Buffer to the smaple and mix by vortexing for 3 seconds.

Add 250 ul ethanol (96-100%) to the lysate, and mix well by pipetting. Do not centrifuge.

Apply sample (700 ul) to the IVT cRNA Cleanup Spin Column. Spin for 15 seconds @ 10,000 x g. Discard flow-through and Collection Tube.

Transfer the spin column to a fresh 2 mL Collection Tube (kit). Pipet 500 ul IVT cRNA Wash Buffer onto the spin column. Spin 15 seconds @ 10,000 x g. Discard flow-through.

Pipet 500 ul 80% EtOH onto spin column, and spin 15 seconds @ 10,000 x g. Discard flow-through.

Open the cap of the spin column and spin 5 minutes @ 18,000 x g to dry the column. Discard flow-through and Collection Tube.

Transfer spin column to a fresh 1.5 mL Collection Tube, and pipet 11 ul of Rnase free Water directly onto the spin column membrane. Spin 1 minute @ 18,000 x g to elute from column.

Pipet 10 ul of Rnase free Water directly onto the spin column membrane. Spin 1 minute @ 18,000 x g to elute.

Fragmenting the cRNA for Target Preparation:

Mix 20 ug cRNA with 8 ul 5X Fragmentation Buffer in 40 ul total volume. The concentration of this sample should be 0.5 ug/ul.

Incubate at 94C for 35 minutes, then quench on ice. Freeze the fragmented sample RNA @ -20C until ready to perform hybridization.

" "1st strand cDNA synthesis:

For each sample, add 2 ul T7-(dT)24 primer @ 50 uM (Affymetrix PN 900375) to 5 ug (up to 8 ul) total RNA. Incubate @ 70C, 10 minutes, quick spin, quench on ice.

Make the following cocktail for 5 reactions using Invitrogen SuperScript Double-Stranded cDNA Synthesis Kit (PN 11917-010): 20 ul 5X 1st Strand cDNA Buffer, 10 ul 0.1 M DTT, and 5 ul 10 mM dNTP mix. Add 7 ul to each reaction. Incubate @ 45 C for 2 minutes; check volume.

To each tube, add 1 ul SuperScript II RT (200 U/ul). Mix by pipetting , incubate @ 45 C for 1 hour in a PCR machine. Total volume should now be 20 ul.

Second strand cDNA synthesis:

Place 1st strand reactions on ice. Spin briefly in microfuge. Make the following cocktail for 5 reactions: 455 ul DEPC-H2O, 150 ul 5X Second Strand Reaction Buffer, 15 ul 10 mM dNTP Mix, 5 ul E.Coli DNA Ligase (10 U/ul), 20 ul E.Coli DNA Polymerase I (10 U/ul), and 5 ul E.Coli RNase H (2 U/ul) for 650 ul total volume. Add 130 ul of the cocktail to each sample.

Gently tap tube to mix. Spin Briefly. Incubate @ 16C for 2 hrs in a PCR machine. Add 2 ul T4 DNA Polymerase (10 U). Mix by pipetting. Incubate @ 16C for 5 minutes in a PCR machine. Add 10 ul 0.5 M EDTA to a fresh 1.5 ml Eppendorf tube and place on ice. Then add the above reaction to this tube to stop the reaction. Proceed to clean up OR store @ -20C for later use.

Cleanup Double-Stranded cDNA using Phase Lock Gels (PLG: Brinkman Instrument, PN 0032007961) and Phenol/Chlorofrom Extraction.

Synthesis of Biotin-Labled cRNA using Enzo Bioarray HighYield RNA Transcript Labeling Kit (Affymetrix PN 900182):

Add 10 ul template ds-cDNA to a new 0.5 mL Rnase-free microfuge tube. Prepare cocktail by adding the following reagents in order: 60 ul DEPC-H2O, 20 ul 10X HYB Reaction Buffer (Vial 1), 20 ul 10X Biotin Labeled Ribonucleotides (Vial 2), 20 ul 10X DTT (Vial 3), 20 ul 10X Rnase Inhibitor Mix (Vial 4), and 10 ul T7 RNA Polymerase (Vial 5) for 150 ul total volume. Then add 30 ul of cocktail to 10 ul of ds-cDNA sample.

Carefully mix the reagents and spin briefly. Incubate @ 37C for 4-5 hrs in a PCR machine, gently mix the contents every 45 minutes during incubation. Proceed to Cleanup OR store @ -20C for future use.

Cleanup Biotin-Labelled cRNA using Qiagen RNeasy kit (PN 74103).

Fragmenting the cRNA for Target Preparation:

Mix 20 ug cRNA and 8 ul 5X Fragmentation Buffer in 40 ul total volume. The concentration of these sample should be 0.5 ug/ul.

Incubate at 94C for 35 minutes, then quench on ice. Freeze the fragmented sample RNA @ -20C until ready to perform hybridization.

" "Hybridization cocktail for single array: 15 ug fragmented labeled cRNA, 5 uL Control Oligonucleotide B2 (component in kit; Affymetrix P/N 900299), 15 uL 20x Eukaryotic Hybridization Controls (component in kit; Affymetrix P/N 900299), 3 uL Herring Sperm DNA (10 mg/mL) (Promega P/N D1811), 3 uL Acetylated BSA (50 mg/mL) (Invitrogen P/N 15561-020), 2x Hybridization Buffer (200 mM MES, 2 M [Na], 40 mM EDTA, 0.02 % Tween 20), and H2O to final volume of 300 uL.

Heat to 95 C for 5 minutes in heat block. Transfer to a 45 C heat block for 5 minutes. Spin 5 minutes at maximum speed in a microcentrifuge.

Fill wetted array with 200 uL of hybridization cocktail. Incubate in Hybridization Oven Model 640 (Affymetrix PN 800138) for 16 h at 45 C at a rotor setting of 50-60 rpm.

" "Microarray Suite v. 5.0.0.032 software (Affymetrix, Inc.) was used to calculate signal values using default settings (Parameters: Gamma 1L, 0.0045; Gamma 1H, 0.0045; Gamma 2L, 0.006; Gamma 2H, 0.006; Perturbation, 1.1; alpha1, 0.05; alpha2, 0.065; tau, 0.015). All data was global scaled to a target intensity of 500. " "Probe set signal values were calculated from CEL files with GeneChip Operating Software v. 1.2.0.003 using the Probe Logarithmic Intensity Error Estimation (PLIER, Affymetrix, Inc.) algorithm using default settings (Probe selection, PM-MM; Background method, none; Optimization, full; Combine replicates, no; Quantile normalization, yes; GM control, 0.15, Attenuation, 0.005, Probe penalty, 0.001, Concentration penalty, 1e-006; Background percent, 0.05; alpha1, 0.05; alpha2, 0.065; tau, 0.015). All data was global scaled to a target intensity of 500.

The PLIER algorithm uses maximum likelihood type estimates in a model-based framework for finding probe expression estimates. It is a multi-chip analysis technique that incorporates information about probe behavior across experiments (affinity estimates) to improve the estimate of concentration for each probe on the array.

An affinity model for the PLIER analysis was constructed from 12 RAE230A arrays on which brain, kidney, liver, or testes RNA, pooled across 8 rats for each of the 3 batches prepared, had been run." "Total RNAs isolated from rat brain (Cat.# 7912), kidney (Cat.# 7926), liver (Cat.# 7910), and testis (Cat.# 7934) were obtained from Ambion (Austin, TX). Total RNAs isolated from rat brain (Cat.# 737001), kidney (Cat.# 737007), liver (Cat.# 737009), and testis (Cat.# 737023) were obtained from Statagene (La Jolla, CA)." "Commercial source." "Mix1 consisted of 5 ug testis RNA, 15 ug liver RNA, 20 ug brain RNA, and 10 ug kidney RNA. 50 ug aliquots of each mixture were frozen at -70C." "Mix2 consisted of 20 ug testis RNA, 10 ug liver RNA, 10 ug brain RNA, and 10 ug kidney RNA. 50 ug aliquots of each mixture were frozen at -70C." "Target labeling of 2 ug total RNA per reaction was performed essentially as described in the CodeLink Expression Bioarrays Automated Target Preparation addendum using the Qiagen BioRobot 9604. CDNA synthesis, cRNA preparation, and cRNA purification were processed in a 96-well format using automated Qiagen Biorobot 9604 procedures. CDNA synthesis were performed using reagents in the CodeLink cDNA Synthesis Kit (PN 320003) supplemented with 10 mM biotin-11-UTP and 10 mM biotin-11-CTP." "Target labeling of 2 ug total RNA per reaction was performed as specified in the manufacturers� protocol in the manual �Codelink Gene Expression System: Manual Labelled cRNA Target Preparation.� CDNA synthesis and in vitro transcription reactions were performed using reagents in the CodeLink Expression Assay Reagent Kit (PN 320012) and 10 mM biotin-11-UTP (PerkinElmer NEL543 or 999-543). Purification of double-stranded cDNA was performed using a QIAquick PCR purification kit (QIAGEN PN 28104 or 28106). Recovery of biotin-labelled cRNA was performed using an Rneasy Mini Kit (QIAGEN PN 74104 or 74106)." "Hybridization and detection was performed as specified in the instruction manual ""Codelink Gene Expression System: Single-Assay Bioarray Hybridization and Detection."" Reagents for fragmentation and hybridization were from the CodeLink Expression Assay Reagent Kit (PN 320012). After fragmentation, 10 ug cRNA was hybridized in a total volume of 260 ul to each CodeLink array for 20 hr at 37C on an orbital shaker.

Post-hybridization, the arrays were washed in 0.75X TNT (1x TNT is 0.1 M Tris-Hcl, pH7.6, 0.15M NaCl, 0.05% Tween-20) at 46C for 1 hr. Detection was performed with a 1:500 dilution of Cy5-Streptavidin (PN 25-0054-31) in TNB (0.1M Tris-Hcl, pH7.6, 0.15M NaCl, 0.5% NEN blocking reagent (PerkinElmer, FP1020)). Arrays were washed four times with 1x TNT, followed by a final rinse in 0.1X SSC/0.05% Tween-20." "Hybridization and detection was performed as specified in the instruction manual ""Codelink Gene Expression System: Single-Assay Bioarray Hybridization and Detection"" and the user guide CodeLink Expression Bioarrays Alexa Fluor 647-Streptavidin Detection Addendum."" Reagents for fragmentation and hybridization were from the CodeLink Expression Assay Reagent Kit (PN 320012). After fragmentation, 10 ug cRNA was hybridized in a total volume of 260 ul to each CodeLink array for 20 hr at 37C on an orbital shaker.

Post-hybridization, the arrays were washed in 0.75X TNT (1x TNT is 0.1 M Tris-Hcl, pH7.6, 0.15M NaCl, 0.05% Tween-20) at 46C for 1 hr. Detection was performed with a 1:500 dilution of Alex Fluor 647-labeled streptavidin (Molecular Probes S-21374) in TNB (0.1M Tris-Hcl, pH7.6, 0.15M NaCl, 0.5% NEN blocking reagent (PerkinElmer, FP1020)). Arrays were washed four times with 1x TNT, followed by a final rinse in 0.05% Tween-20." "Scanning was performed using an Axon 4000B scanner at 10 micron scan resolution, using settings defined in the user manual. Feature extraction was performed using CodeLink Expression Analysis software v2.2.25. Spot intensity was calculated as the average of the pixel signals in the area of interest minus the scaled 2-pixel median background. The intensities were then median normalized by calculating the median of all spots, minus the control probes and blanks, and dividing each spot intensity by the median value." "Scanning was performed using an Axon 4000B scanner at 10 micron scan resolution, using settings defined in the user manual. Feature extraction was performed using CodeLink Expression Analysis software v2.3.2. Spot intensity was calculated as the average of the pixel signals in the area of interest minus the scaled 2-pixel median background. The intensities were then median normalized by calculating the median of all spots, minus the control probes and blanks, and dividing each spot intensity by the median value." "5 ug of total RNA was labeled using the protocol described in Hughes et al. Nature Biotechnol 19: 342-347." "5 ug of total RNA was labeled using the protocol described in Hughes et al. Nature Biotechnol 19: 342-347." "100 ng of total RNA was labeled using the Agilent Low Input RNA Fluorescent Linear Amplification Kit (P/N 5184-3523)." "100 ng of total RNA was labeled using the Agilent Low Input RNA Fluorescent Linear Amplification Kit (P/N 5184-3523)." "500 ng of total RNA was labeled using the Agilent Low Input RNA Fluorescent Linear Amplification Kit (P/N 5184-3523)." "500 ng of total RNA was labeled using the Agilent Low Input RNA Fluorescent Linear Amplification Kit (P/N 5184-3523)." "5 ug of cRNA was hybridized to Agilent G4130A arrays using the protocol described in Hughes et al. Nature Biotechnol 19: 342-347." "0.75 ug labeled cRNA was hybridized per dye channel to Agilent G4130A arrays using Agilent microarray hybridization chambers (G2534A). The arrays were hybridized for 17 hr at 60C at rotor setting 4 in Agilent recommended oven (G2505-80081). Microarrays were washed and dried as specified in the user manual (Agilent 60-mer Oligo Microarray Processing Protocol)." "0.75 ug labeled cRNA was hybridized per dye channel to Agilent G4130A arrays using Agilent microarray hybridization chambers (G2534A). The arrays were hybridized for 17 hr at 60C at rotor setting 5 in Agilent recommended oven (G2505-80081). Microarrays were washed and dried as specified in the user manual (Agilent 60-mer Oligo Microarray Processing Protocol)." "Agilent arrays were scanned using the Agilent DNA microarray scanner (G2565BA) using all default settings. TIFF images were processed with Agilent Feature Extraction software version A.7.4.47 (a prerelease version which is algorithmically identical to v 7.5.1) using default settings. Adjustment for local variations in background signal was performed using a spatial detrending algorithm. To normalize and correct for dye bias, a combined method of linear scaling in each channel followed by LOWESS curve fitting (�Linear&LOWESS� option in version A.7.5) was used. Mix1 and Mix2 signals were calculated from the average of dye-swap replicates. For some applications, this step was followed by normalization of Mix1 to Mix2 using the 10% trimmed mean signal of the kidney-selective probe subset. Features flagged as outliers by the feature extraction software were not removed from the analysis for this study." "Protocol Parameters" "Protocol Software" "SDRF File" "E-TABM-16_sdrf.txt"