Non-radioactive reverse transcriptase/polymerase chain reaction for quantification of myosin heavy chain mRNA isoforms in various rabbit muscles

• Published in: FEBS letters 1993-03, Vol.318 (3), p.253-258
• Main Authors: Peuker, Heidemarie, Pette, Dirk
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Contractile proteins; Digoxigenin; DNA; Fundamental and applied biological sciences. Psychology; Holoproteins; Luminescent Measurements; Male; Muscle fiber type; Muscles - chemistry; Myosin heavy chain mRNA isoform; Myosins - genetics; Number of MHC mRNA molecule; Polymerase Chain Reaction; Proteins; Quantitative reverse transcriptase/polymerase chain reaction; Rabbits; RNA, Messenger - analysis; RNA-Directed DNA Polymerase - metabolism; Tissue Distribution
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(93)80523-W

A method was established for measuring molecule numbers of three different myosin heavy chain (MHC) mRNA isoforms in total RNA preparations. The quantification was based on a combination of primer-directed reverse transcriptase and polymerase chain reactions with 5'-digoxigenin-labeled oligonucleotides, using external standards. The sensitivity of the method allowed the quantitation of mRNA amounts down to the range of 1,000 molecules (detection limit 50 molecules). The numbers determined for eight different rabbit muscles are in the range of 10 3–10 9/μg total RNA. In soleus muscle, the value of 1.11 × 10 9 MHCI mRNA molecules corresponds to approximately 8% of the total mRNA. With reference to myonuclei, this amount corresponds to 1–2 × 10 4 molecules/nucleus. A quantitative comparison of the two fast MHC mRNA isoforms with the distribution of different MHC isoforms at the protein level indicates that one of these two fast sequences is specific to MHCIIb and the other to MHCIId. However, our data point to the existence of additional MHCIId mRNA subtypes.