An mRNA amplification procedure with directional cDNA cloning and strand-specific cRNA synthesis for comprehensive gene expression analysis.

摘要

We developed an integrated system suitable for comprehensive gene expression studies including construction and analysis of cDNA microarrays starting from a small amount of mRNA. We amplified total mRNA first as cDNA mixtures by polymerase chain reaction and then as strand-specific cRNA mixtures by in vitro transcription. These amplified cDNA and cRNA enabled determination of mRNA levels by hybridization analyses such as Southern, Northern, reverse-Northern macroarray, and cDNA microarray analyses, as well as construction of the cDNA library with a unidirectional cDNA insert. By using strand-specific cRNA derived from rat primary-cultured hepatocytes, we detected putative antisense transcripts for the metallothionein gene. cDNA microarray analysis for genes regulated by glucocorticoids and glucagon in the hepatocytes revealed that a number of genes involved in signal transduction and transcriptional regulation were up- or down-regulated. The present system is widely applicable to gene expression analysis with limited amounts of RNA samples.