Schmittgen T D, Zakrajsek B A, Mills A G, Gorn V, Singer M J, Reed M W
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington 99164-6534, USA.
Anal Biochem. 2000 Oct 15;285(2):194-204. doi: 10.1006/abio.2000.4753.
Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3'-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to beta-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r(2) obtained from the linear regression of the first-order mRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.
比较了四种定量逆转录聚合酶链反应（RT-PCR）方法，以评估mRNA形成和衰变的时间进程。在c-fos启动子（fos-glo-myc）控制下转染了人β-珠蛋白开放阅读框/c-myc 3'-非翻译区嵌合基因的小鼠成纤维细胞（NIH 3T3）用于血清诱导转录。在向转染了嵌合基因的血清饥饿成纤维细胞中添加血清后的不同时间，通过定量RT-PCR测量相对于β-肌动蛋白的fos-glo-myc mRNA量。终点法（条带光密度测定和探针杂交）和实时法（SYBR Green和TaqMan）PCR方法均用于检测相同的cDNA。实时法产生了4至5个对数的扩增动态范围，而终点法检测的动态范围为1个对数。实时法和探针杂交法产生的灵敏度水平相当，远高于条带光密度测定法。22次重复PCR反应的变异系数，SYBR Green和TaqMan检测分别为14.2%和24.0%，条带光密度测定和探针杂交法分别为44.9%和45.1%。从一级mRNA衰变图的线性回归获得的r(2)值的排序为SYBR Green>TaqMan>探针杂交>条带光密度测定。实时PCR比终点PCR更精确，动态范围更大。在实时方法中，SYBR Green和TaqMan检测产生了相当的动态范围和灵敏度，而SYBR Green检测比TaqMan检测更精确，产生的衰变图更线性。
