Hollander M C, Fornace A J
Laboratory of Molecular Pharmacology N.C.I., N.I.H., Bethesda, MD 20892.
Biotechniques. 1990 Aug;9(2):174-9.
We have developed an accurate and sensitive method for the quantitation of relative mRNA in RNA samples. This procedure is especially applicable for normalizing numerous RNA samples which are to be analyzed by dot blot hybridization. The method entails hybridization of a polythymidylate probe with RNA bound to filters. The relative hybridization of polythymidylate probe to RNA is proportional to the polyadenylate RNA content of the RNA samples. Hybridization of polythymidylate probe to RNA is not dependent on any cell treatment or growth condition that we have observed, and experimental variation is minimized. Therefore, the relative hybridization to polythymidylate probe is a better method for standardizing the amount of mRNA in RNA samples than is relative hybridization to cDNA probes such as actin or beta 2-microglobulin, whose transcript levels may vary according to cell treatment. Furthermore, since experimental variation and errors are minimized, this procedure is suitable for normalizing RNA samples in which there are only small changes in the levels of particular transcripts.
我们已经开发出一种准确且灵敏的方法，用于定量RNA样本中的相对mRNA。该程序特别适用于对众多通过斑点印迹杂交进行分析的RNA样本进行标准化。该方法需要将聚胸腺嘧啶探针与结合在滤膜上的RNA进行杂交。聚胸腺嘧啶探针与RNA的相对杂交与RNA样本中聚腺苷酸RNA的含量成正比。聚胸腺嘧啶探针与RNA的杂交不依赖于我们所观察到的任何细胞处理或生长条件，并且实验变异被最小化。因此，与聚胸腺嘧啶探针的相对杂交比与肌动蛋白或β2 -微球蛋白等cDNA探针的相对杂交是一种更好的标准化RNA样本中mRNA量的方法，因为后者的转录水平可能会根据细胞处理而变化。此外，由于实验变异和误差被最小化，该程序适用于对特定转录本水平仅有微小变化的RNA样本进行标准化。
