Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC.
Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC.
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通过单管 RT-PCR 和 HPLC 准确、绝对定量测量基因表达。
DOI：
10.1101/gr.5.5.494
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发表时间：
1995
期刊：
影响因子：
7
通讯作者：
Doris,PA
中科院分区：
文献类型：
--
作者：
Hayward-Lester,A;Oefner,PJ;Sabatini,S;Doris,PA
我们报告了一种方法，允许准确的，绝对定量基因表达在一个单一的逆转录酶(RT)-PCR反应。该方法利用新型高效液相色谱（HPLC）技术对竞争性突变RNA pcr的产物进行分析和定量。高效液相色谱技术允许快速，高分辨率的反应产物。在线紫外线检测消除了放射性标签或其他示踪剂的需要。HPLC技术也表明这些竞争反应很容易产生异双相产物。高效液相色谱分析和定量异双相产物的能力是该技术准确性的基础。基因表达的精确测量已经获得了超过四个数量级，实验采用预定数量的特定天然RNA输入已经证明了该系统提供基因表达绝对估计的能力。原生RNA和突变RNA输入之间的巨大差异影响逆转录酶（RT）效率，但不影响PCR扩增效率。然而，RT效率效应的大小是可以估计的，是可重复的，因此可以通过计算的修正因子进行调整。RT效率差异可以通过减少原生RNA和突变RNA之间的序列差异来消除，因此不需要校正因子。应用该技术定量表达的钠，钾- atp酶的α 1亚基在微解剖肾元段被证明。
We report a method that allows accurate, absolute quantification of gene expression in a single reverse transcriptase (RT)-PCR reaction. This method makes use of novel high-performance liquid chromatography (HPLC) technology to resolve and quantify the products of competitive, mutant RNA PCRs. The HPLC technique allows rapid, high resolution of reaction products. On-line UV detection eliminates the need for radiolabel or other tracers. The HPLC technique also demonstrates that these competition reactions readily generate heteroduplex products. The ability of HPLC to resolve and quantify heteroduplex products is fundamental to the accuracy of the technique. Accurate measurements of gene expression have been obtained over four orders of magnitude and experiments employing predetermined quantities of specific native RNA input have demonstrated the ability of the system to provide absolute estimates of gene expression. Large size differences between native and mutant RNA inputs affected reverse transcriptase (RT) efficiency, but not PCR amplification efficiency. However, the magnitude of the RT efficiency effect can be estimated, is reproducible, and can therefore be adjusted by a calculated correction factor. The RT efficiency difference can been eliminated by reduction in the magnitude of the sequence difference between native and mutant RNA so that no correction factor is required. The application of the technique to quantification of expression of the alpha 1 subunit of sodium, potassium-ATPase in microdissected nephron segments is demonstrated.