Showing papers by "Thomas D. Schmittgen published in 2004"
TL;DR: A sensitive, high throughput, real-time PCR assay to monitor the expression of miRNA precursors and proposes that the PCR assay may be scaled up to include all of the 150+ known human miRNA genes and can easily be adaptable to other organisms such as plants, Caenorhabditis elegans and Drosophila.
Abstract: microRNAs (miRNAs) are small, functional, noncoding RNAs. miRNAs are transcribed as long primary transcripts (primary precursors) that are processed to the ~75 nt precursors (pre-miRNAs) by the nuclear enzyme Drosha. The ~22 nt mature miRNA is processed from the pre-miRNA by the RNase III Dicer. The vast majority of published studies to date have used northern blotting to detect the expression of miRNAs. We describe here a sensitive, high throughput, real-time PCR assay to monitor the expression of miRNA precursors. Genespecific primers and reverse transcriptase were used to convert the primary precursors and premiRNAs to cDNA. The expression of 23 miRNA precursors in six human cancer cell lines was assayed using the PCR assay. The miRNA precursors accumulated to different levels when compared with each other or when a single precursor is compared in the various cell lines. The precursor expression profile of three miRNAs determined by the PCR assay was identical to the mature miRNA expression profile determined by northern blotting. We propose that the PCR assay may be scaled up to include all of the 150+ known human miRNA genes and can easily be adaptable to other organisms such as plants, Caenorhabditis elegans and Drosophila.
520 citations