DETERMINING TRANSCRIPT NUMBER USING THE POLYMERASE CHAIN-REACTION - PGK-2, MP2, AND PGK-2 TRANSGENE MESSENGER-RNA LEVELS DURING SPERMATOGENESIS
DETERMINING TRANSCRIPT NUMBER USING THE POLYMERASE CHAIN-REACTION - PGK-2, MP2, AND PGK-2 TRANSGENE MESSENGER-RNA LEVELS DURING SPERMATOGENESIS
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DOI：
10.1093/nar/19.7.1557
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发表时间：
1991-04-11
影响因子：
14.9
通讯作者：
SIMON, MI
中科院分区：
文献类型：
--
作者：
ROBINSON, MO;SIMON, MI
我们描述了一种技术，使用逆转录和聚合酶链反应（PCR），以快速定量的特定mRNA转录物的数量从纳克数量的总细胞RNA。 通过限制循环数和输入RNA的量并通过最小化操作次数来维持输入分子与输出信号的线性。 通过纳入由与实验样品一起运行的连续稀释的体外转录RNA组成的单独标准曲线来确定特异性转录物的绝对水平。 这允许同时快速定量许多样品。 我们应用这种技术来测量磷酸甘油酸激酶2（Pgk-2）转基因在小鼠睾丸发育过程中的表达。 人PGK-2转基因、PGK-2/CAT转基因和内源性mPgk-2基因均显示出相似的表达模式和水平，这与粗线期精母细胞中出现峰值RNA积累的结论一致。 小鼠鱼精蛋白2（mP2）的表达水平比Pgk-2高约10倍，并显示出与单倍体圆形精子细胞中发生的转录起始一致的不同表达模式。
We describe a technique that uses reverse transcription and the polymerase chain reaction (pcr) to rapidly quantitate numbers of specific mRNA transcripts from nanogram quantities of total cellular RNA. Linearity of input molecules to output signal was maintained by limiting the cycle number and the amount of input RNA and by minimizing the number of manipulations. Absolute levels of specific transcripts were determined by the inclusion of a separate standard curve composed of serially diluted in vitro transcribed RNA run alongside the experimental samples. This allowed rapid quantitation of many samples simultaneously. We applied this technique to measuring the expression of phosphoglycerate kinase 2 (Pgk-2) transgenes in the mouse testis during development. A human PGK-2 transgene, a PGK-2/CAT transgene, and the endogenous mPgk-2 gene all displayed similar patterns and levels of expression, consistent with the conclusion that peak RNA accumulation occurs in pachytene spermatocytes. Mouse protamine 2 (mP2) is expressed at a level approximately tenfold higher than Pgk-2 and displays a different pattern of expression consistent with initiation of transcription occuring in haploid round spermatids.