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.