High-throughput RT-PCR analysis of multiple transcripts using a microplate RNA isolation procedure

BioTechniques
S SuF F Craig

Abstract

We have developed a high-throughput, multiplex reverse transcription PCR (RTPCR) assay that is suitable for the analysis of medium-to low-copy cellular RNA transcripts from small numbers of cells (10(4)). High throughput was attained by utilizing microplate-based RNA extraction and RTPCR protocols, followed by PCR product visualization of a multiwelled agarose gel, stained with SYBR Green I dye. The transcriptional assay was unaffected by solvents (dimethyl sulfoxide and methanol) routinely used in high-throughput drug screens at concentrations required for compound solubilization. Furthermore, it has been used successfully for the investigation of differential mRNA expression levels of tumor necrosis factor alpha (TNF-alpha) and Interleukin-1 beta (IL-1 beta) in lipopolysaccharide (LPS)-stimulated THP-1 cells (a human monocytic cell line) and the identification of specific IL-1 beta transcriptional inhibitors.

Citations

Jun 14, 2003·The Journal of Biological Chemistry·Deepika RajeshSteven P Howard
Dec 31, 2004·Lymphatic Research and Biology·Raymond Tabibiazar, Thomas Quertermous
Mar 4, 2014·Tumour Biology : the Journal of the International Society for Oncodevelopmental Biology and Medicine·Deepa Kolaseri KrishnadasKenneth Lucas
Feb 12, 1999·Arteriosclerosis, Thrombosis, and Vascular Biology·P HoustonM Braddock
Jun 16, 2001·International Immunopharmacology·A K MangalamS Naik

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