Enzymatic amplification of myosin heavy-chain mRNA sequences in vitro.
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.
Harbarth
P
P
Max-Planck-Institut für medizinische Forschung, Abteilung Molekulare Biologie, Heidelberg, FRG.
Vosberg
H P
HP
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004262:DNA Restriction Enzymes; D005784:Gene Amplification; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D009928:Organ Specificity; D010446:Peptide Fragments / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D011817:Rabbits; D013045:Species Specificity
D015879:Myosin Subfragments; D010446:Peptide Fragments; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes; D009218:Myosins