Analysis of myosin heavy chain mRNA expression by RT-PCR.
An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.
Wright
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Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
Haddad
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Qin
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Baldwin
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D000818:Animals; D000831:Animals, Newborn / Q000502:physiology; D015152:Blotting, Northern; D007037:Hypothyroidism / Q000378:metabolism / Q000503:physiopathology; D066298:In Vitro Techniques; D007536:Isomerism; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism / Q000502:physiology; D018657:Muscle Fibers, Slow-Twitch / Q000378:metabolism / Q000502:physiology; D009124:Muscle Proteins / Q000096:biosynthesis; D018482:Muscle, Skeletal / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000096:biosynthesis*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley
D009124:Muscle Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D018995:Myosin Heavy Chains