Yin J L, Shackel N A, Zekry A, McGuinness P H, Richards C, Putten K V, McCaughan G W, Eris J M, Bishop G A
Department of Renal Medicine and Centenary Institute, Royal Prince Alfred Hospital and University of Sydney, New South Wales, Australia.
Immunol Cell Biol. 2001 Jun;79(3):213-21. doi: 10.1046/j.1440-1711.2001.01002.x.
Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real-time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA-binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real-time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>107 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (> or =1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low-level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.
实时定量逆转录聚合酶链反应（RT-PCR）是对小样本中细胞因子或生长因子表达进行快速且可重复测量的首选方法。监测实时PCR的荧光检测方法包括用报告染料和淬灭染料标记的荧光探针，如Taqman探针或分子信标，以及双链DNA结合染料SYBR Green I。对一系列人和大鼠细胞因子及生长因子的荧光（Taqman）探针进行了灵敏度测试，并与使用实时荧光监测（PE应用生物系统公司7700型序列检测仪）的SYBR Green I定量分析方法进行了比较。SYBR Green I检测涉及分析PCR产物的解链温度并在最佳温度下测量荧光。荧光探针能够灵敏且可重复地检测表达水平从低（<10个拷贝/反应）到高（>107个拷贝/反应）的靶标。当靶基因以中高水平（≥1000个拷贝/反应）表达时，SYBR Green I能给出可重复的定量结果，但当靶基因低水平表达时，其定量结果并不始终具有可重复性。尽管优化解链温度提高了SYBR Green I检测的特异性，但在我们的实验中，其灵敏度和特异性仍比不上荧光探针。后一种方法是测量低水平基因表达的首选，不过SYBR Green I是定量中高水平表达基因的一种简单且可重复的方法。
