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

S Su, R G Vivier, M C Dickson, N Thomas, M K Kendrick, N M Williamson, J G Anson, J G Houston, F F Craig

Index: Biotechniques 22(6) , 1107-13, (1997)

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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.