Enzymatic amplification of myosin heavy-chain mRNA sequences in vitro

Abstract

We have developed a procedure that detects the presence of mRNA coding for human beta-myosin heavy chain in small amounts of total, unfractionated RNA isolated from heart or skeletal muscle. The protocol is based on the enzymatic amplification in vitro of a selected 106-bp myosin isotype-specific subregion of this mRNA. The method, which is a modification of the so-called "polymerase chain reaction," requires two synthetic oligonucleotide primers (20-mers), reverse transcriptase, and DNA polymerase I (Klenow fragment). Two principle steps are involved: (i) the selected mRNA subregion is converted into a double-stranded cDNA, and (ii) this cDNA is amplified in 22 synthetic cycles. After gel electrophoresis and blotting the amplification product is identified by hybridization with a third oligonucleotide recognizing the region between the two primer annealing sites, and by restriction mapping. Only mRNA from muscle tissue promoted formation of the amplified 106-bp fragment. We estimate that less than 30,000 beta-myosin heavy-chain mRNA molecules are sufficient to produce a signal. The procedure is fast, specific, and very sensitive. It may be used in muscle gene expression studies with small numbers of cells or even in single muscle fibers.