Kagami I, Mizunuma H, Miyamoto S, Ibuki Y, Uchida T
Department of Obstetrics and Gynecology, Gunma University School of Medicine, Japan.
Biochem Biophys Res Commun. 1996 Nov 12;228(2):358-64. doi: 10.1006/bbrc.1996.1666.
A simple and reliable polymerase chain reaction-based method for quantifying human and rat estrogen receptor (ER)-mRNA is described. This method involves co-amplification of cDNA transcribed from sample RNA with an internal standard to reduce tube to tube amplification variations. Human and rat internal standards were synthesized by insertion of a DNA fragment near the midportion of human and rat target ER cDNA molecules. After co-amplification, both products of target ER cDNA and internal standard were separated on agarose gel by electrophoresis and transferred onto a membrane filter. The radioactivity hybridized with 32P-labeled ER cDNA was counted. The ER mRNA content was calculated by linear regression analysis after obtaining a logarithm of ratios between the sample and the internal standard radioactivity. The coefficient of variation of this assay was 18.8%. An absolute amount of ER mRNA in less than 10(4) cultured cells could be measured.
描述了一种基于聚合酶链反应的简单可靠的定量人和大鼠雌激素受体（ER）-mRNA的方法。该方法涉及将样品RNA转录的cDNA与内标共同扩增，以减少管间扩增差异。人和大鼠内标是通过在人和大鼠目标ER cDNA分子中部附近插入一个DNA片段合成的。共同扩增后，目标ER cDNA和内标的产物在琼脂糖凝胶上通过电泳分离并转移到膜滤器上。对与32P标记的ER cDNA杂交的放射性进行计数。在获得样品与内标放射性之比的对数后，通过线性回归分析计算ER mRNA含量。该测定的变异系数为18.8%。可以测量少于10(4)个培养细胞中ER mRNA的绝对量。
