The stress kit: a new method based on competitive reverse transcriptase-polymerase chain reaction to quantify the expression of human alpha B-crystallin, Hsp27, and Hsp60

We describe a reverse transcriptase-polymerase chain reaction method for th e semiquantitative detection of mRNAs encoding the human heat shock protein s alpha B-crystallin, Hsp27, and Hsp60. The method involves the coamplifica tion of cellular mRNA-derived cDNA with a dilution series of a competitor f ragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be sli ghtly smaller to differentiate between the target and the standard on elect rophoretic separation. Initial cDNA quantitations can be corrected for poss ible variations during cDNA synthesis by standardizing to the levels of bet a-actin-encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the expon ential and the nonexponential phase of amplification. Furthermore, we illus trate the utility of this technique by quantifying increased expression of alpha B-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shoc k.