Benoff S, Nadal-Ginard B
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1853-7. doi: 10.1073/pnas.76.4.1853.
The mRNA for rat muscle myosin heavy chain (MHC) was isolated from L6E9 myotubes by two rounds of sucrose density gradient centrifugation followed by fractionation on an agarose/polyacrylamide gel. The purity of the mRNA isolated was determined by translation in vitro, peptide analysis of the in vitro product and comparison with authentic MHC, analysis of the kinetics of hybridization with cDNA prepared with this RNA, and titration analysis of total cytoplasmic RNA from muscle and nonmuscle sources. By using the MHC cDNA as probe of myogenic differentiation, it was observed that the level of cytoplasmic MHC mRNA increased approximately 200-fold as the dividing myoblast differentiated into the fused myotube. Titration analysis of RNAs fractionated by oligo(dT)-cellulose chromatography indicated that the majority of the increase occurred in that RNA population that failed to bind to an oligo(dT)-cellulose column.
通过两轮蔗糖密度梯度离心，随后在琼脂糖/聚丙烯酰胺凝胶上进行分级分离，从L6E9肌管中分离出大鼠肌肉肌球蛋白重链（MHC）的mRNA。通过体外翻译、体外产物的肽分析以及与真实MHC的比较、与用该RNA制备的cDNA杂交动力学分析，以及对来自肌肉和非肌肉来源的总细胞质RNA的滴定分析，来确定分离的mRNA的纯度。通过使用MHC cDNA作为成肌分化的探针，观察到随着分裂的成肌细胞分化为融合的肌管，细胞质MHC mRNA水平增加了约200倍。对通过寡聚（dT）-纤维素色谱法分级分离的RNA进行滴定分析表明，增加的大部分发生在未与寡聚（dT）-纤维素柱结合的RNA群体中。
