CONSTRUCTING POLYCOMPETITOR CDNAS FOR QUANTITATIVE PCR
CONSTRUCTING POLYCOMPETITOR CDNAS FOR QUANTITATIVE PCR
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DOI：
10.1016/0022-1759(93)90104-f
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发表时间：
1993-09-27
影响因子：
2.2
通讯作者：
LOCKSLEY, RM
中科院分区：
文献类型：
--
作者：
REINER, SL;ZHENG, SC;LOCKSLEY, RM
使用逆转录结合聚合酶链反应分析mRNA水平为研究细胞免疫学中的细胞因子调节提供了有力的工具。我们报告了一种新的方法克隆竞争的cDNA，是快速，有效和廉价的。通过串联连接多个竞争者cDNA，可以产生多竞争者构建体，其允许使用单一试剂进行单独的PCR测定。测定可以在细胞培养物或组织的微小样品上进行，并且可以在常规凝胶电泳后可靠地定量，而无需使用光密度测定法或标记核苷酸。该技术的实用性在于能够生产相对便宜的定制试剂，其使用简单并且允许以快速和方便的方式灵敏地测定基因表达。这种方法应该允许生物学的许多领域的研究人员容易地定量范围广泛的重要调节分子。
Analysis of mRNA levels using reverse transcription coupled with the polymerase chain reaction provides a powerful tool for studying cytokine regulation in cellular immunology. We report a novel method for cloning competitor cDNAs that is rapid, efficient and inexpensive. By linking multiple competitor cDNAs in tandem, polycompetitor constructs can be created that allow the use of a single reagent for individual PCR assays. Assays can be performed on minute samples of cell culture or tissue and can be reliably quantitated after routine gel electrophoresis without the use of densitometry or labeled nucleotides. The utility of this technique lies in the ability to produce a relatively inexpensive customized reagent that is simple to use and that allows for sensitive determinations of gene expression in a rapid and convenient manner. This method should allow investigators in many areas of biology to easily quantitate a broad range of important regulatory molecules.