A novel method for real time quantitative RT PCR
A novel method for real time quantitative RT PCR
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DOI：
10.1101/gr.6.10.995
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发表时间：
1996-10-01
期刊：
影响因子：
7
通讯作者：
Williams, PM
中科院分区：
文献类型：
--
作者：
Gibson, UEM;Heid, CA;Williams, PM
已经开发了一种使用实时检测和5'核酸酶测定的定量逆转录酶聚合酶链反应（QC RT-PCR）的新型方法。囊性纤维化跨膜转置调节剂（CFTR）靶mRNA被实时反转录，放大，检测和定量。设计了荧光探针来检测CFTR扩增子。使用分析热循环仪，在PCR扩增过程中监测6羧基 - 氟洛雷斯素报告蛋白报告荧光发射的相对增加。包含与CFTR扩增子相同的引物序列的内部控制模板，但内部序列已被设计为对照。具有报告基因荧光染料Tetrachloro-6-羧基 - 氟氟乙烯素的内部对照探针的设计，可与内部控制扩增子杂交。内部控制模板置于每个反应管中，用于定量分析CFTR mRNA。此方法为QC RT-PCR提供了方便且高通量的格式。
A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.