Genome-wide identification and analysis of mRNA expression in MEFs, ESCs, and iPSCs

Microarray analysis of mRNARNA was prepared from ESCs (CGR8 and CJ7), MEFs, and iPSCs with a PureLink RNA Mini Kit (Life Technologies). Initial sample quality control was performed with a Nanodrop 8000 (Thermo Scientific). RNA samples with an optical density (OD) 260/280 ratio and an OD 260/230 ratio of 1.8 or higher were used. Samples with an RNA Integrity Number of 7.5 or greater measured with a LabChip Caliper GX (Perkin Elmer) were applied to an Illumina TotalPrep-96 RNA Amplification Kit (Life Technologies) to generate biotinylated and amplified RNA. With this kit, 300 ng of total RNA was first processed in a reverse transcription reaction. The cDNA then underwent second-strand synthesis and cleanup to serve as a template for in vitro transcription, which generated biotinylated antisense RNA copies of each mRNA. Samples went through another round of quality control with the Nanodrop 8000 and were applied to Illumina MouseWG-6 v2.0 Beadchips (#BD-201-0202). After overnight hybridization, the Beadchips were washed, stained, and scanned using an Illumina iScan Beadarray Reader. The obtained data were analyzed with an Illumina Genome Studio.

12

Hirai H, Firpo M, Kikyo N