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Department of Physiology and Biophysics, University of California, Irvine, California 92697
Received 6 March 1997; accepted in final form 3 June 1997.
Wright, Carola, Fadia Haddad, Anqi X. Qin, and Kenneth M. Baldwin. Analysis of myosin heavy chain mRNA
expression by RT-PCR. J. Appl.
Physiol. 83(4): 1389-1396, 1997.
An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC)
mRNA expression in rodent skeletal muscle. Only 2 µg 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.
skeletal muscle; Northern blot; semiquantitative competitive reverse transcriptase-polymerase chain reaction; Sprague-Dawley rats; messenger ribonucleic acid
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