Sanchez H, Chapot R, Banzet S, Koulmann N, Birot O, Bigard A X, Peinnequin A
Département des facteurs humains, Centre de Recherches du Service de Santé des Armées, BP87, 38702 La Tronche Cedex, France.
Biochem Biophys Res Commun. 2006 Feb 3;340(1):165-74. doi: 10.1016/j.bbrc.2005.11.172. Epub 2005 Dec 9.
A real-time RT-PCR assay using newly designed primers was developed to analyze developmental and adult MHC mRNA expression both in skeletal muscles and single fibers. Only 4 ng of total RNA was necessary for the analysis of the relative mRNA expression of MHC genes. Different validation steps were realized concerning both specificity and sensitivity of each primer set, and linearity and efficiency of each real-time PCR amplification. Then, quantification of MHC mRNA in neonatal and adult muscles as well as in single fibers was done by the deltaC(T) method, with CycA gene as the reference gene. Due to a higher sensitivity than that of a competitive PCR method, we demonstrated that this assay is suitable to study very low level of MHC mRNA expression as developmental MHC in adult muscle and to quantify mRNA from very small samples.
利用新设计的引物开发了一种实时逆转录聚合酶链反应（RT-PCR）检测方法，用于分析骨骼肌和单根肌纤维中发育阶段和成年期主要组织相容性复合体（MHC）mRNA的表达。分析MHC基因的相对mRNA表达仅需4 ng总RNA。针对每个引物组的特异性和敏感性以及每个实时PCR扩增的线性和效率，都进行了不同的验证步骤。然后，采用ΔC(T)法，以CycA基因作为参照基因，对新生肌肉和成年肌肉以及单根肌纤维中的MHC mRNA进行定量。由于该检测方法比竞争性PCR方法具有更高的灵敏度，我们证明该检测方法适用于研究成年肌肉中发育阶段的MHC这种极低水平的MHC mRNA表达，以及对非常小的样本中的mRNA进行定量。
