Real‐time reverse transcriptase–polymerase chain reaction (RT–PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I
Real‐time reverse transcriptase–polymerase chain reaction (RT–PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I
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DOI：
10.1046/j.1440-1711.2001.01002.x
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发表时间：
2001-06
影响因子：
4
通讯作者：
Jianlin Yin;N. Shackel;A. Zekry;P. H. McGuinness;C. Richards;Karien van der Putten;G. McCaughan;J. Eris;G. Bishop
中科院分区：
文献类型：
--
作者：
Jianlin Yin;N. Shackel;A. Zekry;P. H. McGuinness;C. Richards;Karien van der Putten;G. McCaughan;J. Eris;G. Bishop
真实的实时定量逆转录酶-聚合酶链反应（RT-PCR）是快速、可重复测量小样本中细胞因子或生长因子表达的首选方法。用于监测真实的-时间PCR的荧光检测方法包括用报告和猝灭染料标记的荧光探针，例如Taqman探针或分子生物学和dsDNA结合染料SYBR绿色I。检测了一系列人和大鼠细胞因子和生长因子的荧光（Taqman）探针的灵敏度，并与使用真实的荧光监测（PE Applied Biosystems Model 7700序列检测器）的SYBR绿色I定量测定进行了比较。SYBR绿色I检测包括分析PCR产物的解链温度和在最佳温度下测量荧光。荧光探针提供了从低（107个拷贝/反应）表达的靶标的灵敏和可重复检测。当靶基因以中等至高水平表达（≥1000拷贝/反应）时，SYBR绿色I给出了可重现的定量，但当靶基因以低水平表达时，不能给出一致的可重现定量。尽管解链温度的优化提高了SYBR绿色I检测的特异性，但在我们手中，它并不等同于荧光探针的可重复灵敏度和特异性。后一种方法是测量低水平基因表达的首选，尽管SYBR绿色I是一种简单且可重复的方法来量化中高水平表达的基因。
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 (107 copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (≥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.