Luo X Z, Stevens S E
University of Memphis, TN 38152, USA.
Biotechniques. 1997 Nov;23(5):904-6, 908, 910. doi: 10.2144/97235st08.
Isolation of full-length mRNA without degradation is critical in the study of in vivo gene regulation and transcription, cDNA synthesis and reverse transcription (RT)-PCR. It is particularly difficult to isolate full-length mRNA from thermophiles, which have higher turnover rates of mRNA degradation. Mastigocladus laminosus is a thermophilic heterocystous cyanobacterium. The assay of M. laminosus cell lysates showed that RNase activity was high and was resistant to the conventional guanidine thiocyanate and 2-mercaptoethanol denaturation methods. The mRNA isolated by several conventional methods was completely degraded. A method was developed to purify full-length mRNA by a combination of fast cooling, vanadyl-ribonucleoside-complex inhibition, phenol-chloroform-isoamyl alcohol extraction, lithium chloride precipitation and the lysing of cells with the French Press. This method produced high-quality, full-length mRNA in high yield. Purified mRNA was suitable for Northern blotting, cDNA synthesis and RT-PCR. This method could be applicable to other thermophiles in which the RNase activity is high and/or is resistant to guanidine thiocyanate.
分离未降解的全长mRNA在体内基因调控与转录、cDNA合成及逆转录（RT）-PCR研究中至关重要。从嗜热菌中分离全长mRNA尤为困难，因为嗜热菌的mRNA降解周转率更高。层理鞭枝藻是一种嗜热的异形胞蓝细菌。层理鞭枝藻细胞裂解物的检测表明，其核糖核酸酶活性很高，并且对传统的硫氰酸胍和2-巯基乙醇变性方法具有抗性。通过几种传统方法分离的mRNA完全降解。开发了一种通过快速冷却、钒核糖核苷复合物抑制、苯酚-氯仿-异戊醇提取、氯化锂沉淀以及用法国压榨机裂解细胞相结合的方法来纯化全长mRNA。该方法能以高产率产生高质量的全长mRNA。纯化的mRNA适用于Northern印迹、cDNA合成及RT-PCR。该方法可应用于核糖核酸酶活性高和/或对硫氰酸胍具有抗性的其他嗜热菌。
