Multiplex standardized RT-PCR for expression analysis of many genes in small samples
Multiplex standardized RT-PCR for expression analysis of many genes in small samples
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DOI：
10.1016/s0006-291x(02)00243-7
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发表时间：
2002-04-26
影响因子：
3.1
通讯作者：
Willey, JC
中科院分区：
文献类型：
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作者：
Crawford, EL;Warner, KA;Willey, JC
标准化RT-PCR(START-PCR)使基因表达的数值量化以及实验室内和实验室间的比较成为可能。在Stratagene Universal Reference RNA上进行了两轮扩增的多重START-PCR。在第一轮中，扩增了多达96个基因的cDNA、竞争性模板(CT)混合物和引物，扩增周期数各不相同。接下来，第一轮的产品被稀释。与一个基因的引物结合，再扩增35个循环。没有添加额外的cDNAs或CT混合物。Uniplex和Mplex Start-PCR获得的表达值高度相关(R=0.993，p<；0.001)，第一轮扩增的产物可以稀释100,000倍，第二轮扩增后仍可定量。因此，使用多重START-PCR，在相同数量的cDNA量中测量了96个基因，通常用于测量一个基因的单链START-PCR。应用多重启动-聚合酶链式反应(MSTR)对1例肺癌细针活检组织中的18个基因进行了检测。(C)2002年埃尔塞维尔科学公司(美国)。版权所有。
Standardized RT-PCR (StaRT-PCR) enables numerical quantification as well as intra- and inter-laboratory comparison of gene expression. Multiplex StaRT-PCR, using two rounds of amplification, was conducted on Stratagene Universal Reference RNA. In the first round, cDNA, competitive template (CT) mix, and primers for up to 96 genes were amplified for varying numbers of cycles. Next, products from round one were diluted. combined with primers for one gene, and amplified for an additional 35 cycles. No additional cDNA or CT mix was added. Expression values obtained by uniplex and multiplex StaRT-PCRs were highly correlated (R = 0.993, p < 0.001), Products from round one could be diluted as much as 100,000-fold and still be quantified following round two amplification. Thus, using multiplex StaRT-PCR, 96 genes were measured in the same amount of cDNA typically used to measure one gene with uniplex StaRT-PCR. Multiplex StaRT-PCR was also used to measure 18 genes in the fine needle biopsy of a primary lung carcinoma. (C) 2002 Elsevier Science (USA). All rights reserved.