Non‐radioactive reverse transcriptase/polymerase chain reaction for quantification of myosin heavy chain mRNA isoforms in various rabbit muscles
Non‐radioactive reverse transcriptase/polymerase chain reaction for quantification of myosin heavy chain mRNA isoforms in various rabbit muscles
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DOI：
10.1016/0014-5793(93)80523-w
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发表时间：
1993-03
期刊：
影响因子：
3.5
通讯作者：
H. Peuker;D. Pette
中科院分区：
文献类型：
--
作者：
H. Peuker;D. Pette
建立了一种测定总RNA制备物中三种不同肌球蛋白重链（MHC）mRNA亚型分子数的方法。定量是基于引物导向的逆转录酶和聚合酶链反应与5 '-地高辛标记的寡核苷酸的组合，使用外部标准。该方法的灵敏度允许定量mRNA量低至1，000个分子的范围（检测限50个分子）。八种不同兔肌肉的测定值在103-109/μg总RNA范围内。在比目鱼肌中，1.11 × 109 MHCI mRNA分子的值相当于总mRNA的约8%。对于肌核，该量相当于1-2 × 104个分子/核。两种快速MHC mRNA同种型与不同MHC同种型在蛋白质水平上的分布的定量比较表明，这两种快速序列中的一种对MHCIIb特异，另一种对MHCIId特异。然而，我们的数据表明存在额外的MHCIId mRNA亚型。
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 103–109/μg total RNA. In soleus muscle, the value of 1.11 × 109MHCI mRNA molecules corresponds to approximately 8% of the total mRNA. With reference to myonuclei, this amount corresponds to 1–2 × 104molecules/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.