HIGH-THROUGHPUT RT-PCR ANALYSIS OF MULTIPLE TRANSCRIPTS USING A MICROPLATE RNA ISOLATION PROCEDURE

We have developed a high-throughput, multiplex reverse transcription P CR (RT-PCR) assay that is suitable for the analysis of medium- to low- copy cellular RNA transcripts from small numbers of cells (10(4)). Hig h throughput was attained by utilizing microplate-based RNA extraction and RT-PCR protocols, followed by PCR product visualization of a mult iwelled agarose gel, stained with SYBR(R) Green I dye. The transcripti onal assay was unaffected by solvents (dimethyl sulfoxide and methanol ) routinely used in high-throughput drug screens at concentrations req uired for compound solubilization. Furthermore, it has been used succe ssfully for the investigation of differential mRNA expression levels o f tumor necrosis factor alpha (TNF-alpha) and Interleukin-1 beta (IL-1 beta) in lipopolysaccharide (LPS)-stimulated THP-I cells (a human mon ocytic cell fine) and the identification of specific IL-1 beta transcr iptional inhibitors.