Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR
Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR
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DOI：
10.1023/a:1005658330108
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发表时间：
2001-02-01
影响因子：
2.7
通讯作者：
Hageleit, M
中科院分区：
文献类型：
--
作者：
Pfaffl, MW;Hageleit, M
逆转录（RT）和聚合酶链反应（PCR）是分析极低丰度mRNA的首选技术。使用SYBR Green I检测的实时RT-PCR结合了简单和必要的准确性，能够产生可靠和快速的结果。为了在RT和real-time PCR中获得较高的准确性和可靠性，需要高度定义的校准曲线。我们基于重组IGF-1 RNA （recRNA）或重组IGF-1 DNA （recDNA）校准曲线，在LightCycler中开发、优化并验证了胰岛素样生长因子-1 (IGF-1) RT-PCR。在此基础上，确定了这些外部标准化定量系统的限、准确性和变异，并与肝脏总RNA的天然RT-PCR进行了比较。为了评价和优化recRNA的cDNA合成速率，测试了几种RNA背景。我们得出结论，使用recDNA的外部校准曲线比recRNA校准模型更适合mRNA的定量。与recRNA校准曲线相比，该模型具有更高的灵敏度、更大的定量范围、更高的重现性和更稳定的稳定性。
Reverse transcription (RT) followed by polymerase chain reaction (PCR) is the technique of choice for analysing mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection combines the ease and necessary exactness to be able to produce reliable as well as rapid results. To obtain high accuracy and reliability in RT and real-time PCR a highly defined calibration curve is needed. We have developed, optimised and validated an Insulin-like growth factor-1 (IGF-1) RT-PCR in the LightCycler, based on either a recombinant IGF-1 RNA (recRNA) or a recombinant IGF-1 DNA (recDNA) calibration curve. Above that, the limits, accuracy and variation of these externally standardised quantification systems were determined and compared with a native RT-PCR from liver total RNA. For the evaluation and optimisation of cDNA synthesis rate of recRNA several RNA backgrounds were tested. We conclude that external calibration curve using recDNA is a better model for the quantification of mRNA than the recRNA calibration model. This model showed higher sensitivity, exhibit a larger quantification range, had a higher reproducibility, and is more stable than the recRNA calibration curve.