GenomeTiling

Samples were purchased from Clontech as mRNA. Hybridization material was generated through a random-priming amplification procedure (RP-AMP) using primers with a random sequence at the 3' end and a fixed motif at the 5' end. shDNP256 (first strand synthesis): 5'- TAG ATG CTG TTG NNN NNN NNN -3' shT7N9 (second strand synthesis): 5'- ACT ATA GGG AGA NNN NNN NNN -3' 1.5 g of mRNA was reverse-transcribed with Superscript II and the DP256 random primer for 20 minutes at 42 C (10 mM DTT, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 5 U/ l Superscript II). The RNA was degraded with the addition of 20 l volume of 0.5N sodium hydroxide and 0.25 M EDTA for 20 minutes at 65 C. The single stranded cDNA was purified using a commercial kit (Qiagen Qiaquick). The resulting product of single stranded cDNA was placed in its entirety in a second strand reaction. Second strand synthesis reactions utilized shT7N9 random primer and the Klenow fragment of DNA polymerase utilizing standard reaction conditions (37 C for 60 minutes, 0.2 mM DTT, 2.1 mM Tris-HCl pH 7.9, 2.1 mM MgCl2, 10.7 mM NaCl, 1.07 mM dNTPs, 0.1U/ l Klenow), followed by another Qiaquick purification. Multiple polymerase chain reactions were run using 0.15 g of double stranded DNA and standard reaction conditions. Amplification was achieved using 10 cycles of PCR with the DP256 and T7 primers (20 mM Tris-HCl pH 8.4, 50 mM KCl, 0.01 mM dNTPs, 1.5 mM MgCl2, 0.01 U/ l Taq Polymerase) DP256: 5'- GTT CGA GAC CTC TAG ATG CTG TTG -3' T7: 5'- AA TTA ATA CGA CTC ACT ATA GGG AGA -3' followed by Qiaquick purification. Further amplification was achieved using in vitro transcription reactions with X0.5 g dsDNA and T7 RNA Polymerase (7.5 mM DTT, 40 mM Tris-HCl pH 7.5, 14.25 mM MgCl2, 10 mM NaCl, 2 mM Spermidine, 125 U/ml RNAguard, 2.5 mM dNTPs, 15 U/ml IPPase, 25kU/ml T7 Polymerase) for 16 hours at 42 C. The cRNA was purified (RNeasy) and reverse transcribed using Superscript and random 9-mers and amino-allyl dUTP (42 C for 20 minutes, 10 mM DTT, 50mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 0.5 mM aa-dUTP, 5 U/ l Superscript II). The cDNA for each channel was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 1M sodium bicarbonate, pH 9.0, followed by quenching with 4.0 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were purified with a QIAquick PCR purification kit and the percentage dye incorporation and total cDNA yield was determined spectrophotometrically. Cy5 and Cy3 labelled cDNAs were combined and added to 2 mls 30% formamide with Herring Sperm DNA for hybridization to the microarray. Further details can be found in Castle et al., Genome Biol 2003;4(10):R66,