Affymetrix GeneChip Rat Expression Array RAE230A [RAE230A]

Comparative analysis of methods for gene transcription profiling data derived from different microarray technologies in rat and mouse models of diabetes.

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Effects of hyperglycaemia and genetic background differences on gene expression in rats

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Transcription profiling of three strains of rat that are normoglycaemic or hyperglycaemic

Effects of hyperglycaemia and genetic background differences on gene expression in rats


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4_450

Title: Affymetrix EukGE-WS2v4_450 Hybridization. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-34508

Total RNA was extracted twice using Trizol reagent (Invitrogen Life Technologies, Paisley, UK) according to the manufacturer’s instructions and cleaned with RNeasy columns (Qiagen Ltd., Crawley, UK). RNA concentration and integrity were assessed using a spectrophotometer and the Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany). First strand cDNA was synthesized using 14µg of each individual total RNA samples (no pooling). For a 20µl reaction, 1µl of 100pmol T7-Oligo(dT)24 primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3') was used. A reaction containing 7µl of a master mix (4µl of 5X first strand buffer; 2µl of 0.1M DTT and 1µl of 10mM dNTPs) was incubated for 2min at 42C, before adding 2µl of SuperScript II Reverse Transcriptase (Invitrogen Life Technologies, Paisley, UK) (400 Units total). Incubation was carried out at 42C for a further 60min, after which the reaction was inactivated on ice before proceeding to second strand cDNA synthesis. Double-stranded DNA was synthesized by adding the first strand cDNA reaction to a master mix (30µl of 5X second strand buffer; 3µl of 10mM dNTPs; 1µl of DNA Ligase (10 Units); 4µl of DNA Polymerase I (40 Units); 1µl of RNase H (2 Units); 91µl of distilled water).The reaction was incubated at 16C for 2h and inactivated on ice before cleanup. Double-stranded cDNA were purified using reagents of the GeneChipÒ Sample Cleanup Module (Affymetrix Ltd., High Wycombe, UK). A 600µl aliquot of cDNA binding buffer was added to the reaction. A 500µl aliquot was applied to the cleanup spin column and centrifuged at 10,000rpm for 1min. The flow-through was discarded and the spin column reloaded with the remaining volume of cDNA. The column was centrifuged at 10,000rpm for 1min. After transferring the column into a new collection tube, 750µl of cDNA wash buffer were added. The column was spun at 10,000rpm for 1min, and the flow-through removed. Centrifugation of the tubes was repeated with open cap in order to completely dry the membrane. Purified double-stranded cDNA was eluted in a fresh tube by placing 14µl of a cDNA elution buffer onto the membrane. The column was incubated for 1min at room temperature and double-stranded cDNA was eluted by spinning at 12,000rpm for 1min.

P-MEXP-34509

Purified cDNA was used as template for in vitro transcription reaction for the synthesis of biotinylated cRNA, using the Enzo BioArrayTM High YieldTM RNA Transcript Labeling kit (Enzo Life Sciences, Inc., Farmingdale, NY). An aliquot of 12µl of double-stranded cDNA was mixed with 28µl of a master mix (10µl nuclease-free distilled water; 4µl 10X high yield reaction buffer; 4µl biotin-labelled ribonucleotides; 4µl DTT; 4µl RNase inhibitor mix; 2µl T7 RNA Polymerase) and incubated at 37oC for 5h. Biotin-labelled cRNA was purified to remove unincorporated biotinylated ribonucleotides using reagents of the GeneChipÒ Sample Cleanup Module (Affymetrix Ltd., High Wycombe, UK). cRNA binding buffer (350µl) and distilled water (60µl) were added to the in vitro transcription reaction before adding 100% ethanol (250µl). The sample was then applied to a cRNA cleanup column and spun at 10,000rpm for 15sec. After transferring the spin column into a fresh collection tube, 500µl of cRNA wash buffer were added and the column was spun at 10,000rpm for 15sec. The flow-through was discarded, 500µl of 80% ethanol were added onto the column, which was spun again at 10,000rpm for 15sec. After discarding the flow-through, the column was centrifuged at 12,000rpm for 5min in order to completely dry the membrane. Purified biotin-labelled cRNA was finally eluted twice into a new collection tube with a total of 21µl of RNase-free water, and spinning at 12,000rpm for 1min. Purified biotin-labelled cRNA was quantified and the quality/integrity of the cRNA was further assessed using the Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany). Biotinylated cRNA (15µg) was fragmented at 95°C for 35min in 13µl of 5X fragmentation buffer (supplied with the AffymetrixÒ GeneChipÒ Sample Cleanup Module), made up to 65µl with distilled water. After incubation, 235µl of a hybridisation cocktail (150µl 2X hybridisation buffer; 5µl 3nM control oligonucleotide B2 pre-heated at 65°C for 5min; 15µl 20X eukaryotic hybridisation controls (BioB, BioC, BioD, cre) pre-heated at 65°C for 5min; 3µl 10mg/ml herring sperm DNA; 3µl 50mg/ml acetylated BSA; 59µl RNase-free distilled water) were added to the cRNA fragmentation reaction. Prior to hybridisation, fragmented biotinylated cRNA mixtures were heated at 99°C for 5min, then cooled at 45oC for 5min, and finally spun at 12,000rpm for 5min.

P-MEXP-34562

Spontaneously diabetic Goto-Kakizaki (GK/Ox) inbred rats were from the colony initiated in Oxford in 1995 using breeders from the colony maintained in Paris (CNRS URA 307, Paris, France). Inbred Brown-Norway (BN) rats were obtained from a commercial supplier (Charles River Laboratories, Margate, UK). Wistar-Kyoto (WKY) rats were obtained from a commercial supplier (Harlan, Bicester, UK). Rats were weaned at 21-24 days of age and maintained in groups of 5 rats in standard conditions in open top cages according to UK Home Office regulations. Rats had free access to water and standard laboratory chow pellets (B&K Universal, Hull, UK) and were maintained on a 12-h light and dark cycle with light off at 8pm. Temperature was maintained at 20-22 °C. Three month old male rats were killed by CO2 asphyxiation between 9 and 10 a.m. after an overnight fast.

P-MEXP-34563

Organs were rapidly dissected out and immediately snap frozen in liquid nitrogen and stored at -80°C until use.

Transcription profiling of three strains of rat that are normoglycaemic or hyperglycaemic

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