Affymetrix GeneChip Rat Expression Array RAE230A [RAE230A]

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To systemically explore how RNA quality affects microarray assay results, a set of rat liver RNA samples with a progressive change in RNA quality was generated either by thawing frozen tissue or by ex vivo incubation of fresh tissue.

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Transcription profiling of fresh and frozen rat liver RNA samples to explore how RNA quality affects microarray results

Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA

To systemically explore how RNA quality affects microarray assay results, a set of rat liver RNA samples with a progressive change in RNA quality was generated either by thawing frozen tissue or by ex vivo incubation of fresh tissue.


P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-50537

After euthanasia by carbon dioxide inhalation, whole livers were removed and placed in Petri dishes containing sterile phosphate buffered saline (PBS). Livers were briefly rinsed with PBS to remove blood. Liver sections were prepared by removing a 2 cm square section from the left lateral lobe and further sectioning it into 12-16 equal pieces. Each time course study used a single liver lobe from a unique animal. Incubations at 37°C were performed in a water bath and terminated by addition of RNAlater.  After the samples were incubated in RNAlater overnight, RNA was isolated using Qiagen Midi kits (Qiagen, Valencia, CA).

P-MEXP-50538

After euthanasia by carbon dioxide inhalation, whole livers were removed and placed in Petri dishes containing sterile phosphate buffered saline (PBS).  Livers were briefly rinsed with PBS to remove blood.  Liver sections were prepared by removing a 2 cm square section from the left lateral lobe and further sectioning it into 12-16 equal pieces.  Each time course study used a single liver lobe from a unique animal.  To generate samples in which RNA was degraded after tissue thawing, liver sections were snap frozen in a dry ice/ethanol slurry and stored at -70°C.  Random tissue sections were thawed at room temperature for various intervals.  At the end of each incubation period, samples were homogenized in Qiagen RLT buffer and processed following the Qiagen Midi kit protocol.

P-MEXP-50715

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. 

P-MEXP-50735

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.

P-MEXP-50736

All tissue RNAs were isolated using a Tempest rotor-stator homogenizer (VirTis, Gardiner, NY) and Qiagen Midi kits (Qiagen, Chatsworth, CA), following the manufacturer's protocol. RNA and cRNA yields were quantitated on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).  All samples had 260/280 ratios > 2.0.  RNA integrity was characterized by measuring the 28s/18s rRNA ratio and RIN on an Agilent 2100 Bioanalyzer (Santa Clara, CA).

P-MEXP-50737

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.

P-MEXP-50738

GeneChips were scanned on a GeneArray 2500 scanner using MicroArray Suite v. 5.0.0.032 and default scanner settings.

P-MEXP-52492

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.

Transcription profiling of fresh and frozen rat liver RNA samples to explore how RNA quality affects microarray results

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