A method for the quantitative analysis of human heat shock gene expressionusing a multiplex RT-PCR assay

A quantitative multiplex RT-PCR assay is described to measure the levels of messenger RNAs for eight human genes encoding the heat shock proteins (HSP ) and molecular chaperones hsp90 alpha,, hsp90 beta P, hsp70, hsc70, mtHsp7 5, Grp78 (BiP), hsp60 and hsp27. The basis of this assay is reverse transcr iption of total RNA isolated from human cells followed by amplification wit h PCR. By the careful selection of pairs of oligonucleotide primers corresp onding to unique regions of each heat shock gene, selectivity can be attain ed such that messenger RNAs of multiple heat shock genes can be analyzed si multaneously in a single reaction. This method provides both the absolute a nd relative levels of each heat shock message by including in the reaction, reference control RNAs corresponding to in vitro transcripts of heat shock gene plasmids carrying small internal deletions.