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首页> 美国卫生研究院文献>Journal of Biomolecular Techniques : JBT >Two-Step Versus One-Step RNA-to-CT™ 2-Step and One-Step RNA-to-CT™ 1-Step: Validity Sensitivity and Efficiency
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Two-Step Versus One-Step RNA-to-CT™ 2-Step and One-Step RNA-to-CT™ 1-Step: Validity Sensitivity and Efficiency

机译：两步与一步RNA-to-CT™2步和一步RNA-to-CT™1步：有效性敏感性和效率

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摘要

Quantitative RT-PCR can be carried out as a one- or a two-step reaction. However, the choice of method raises controversy from the perspective of the researcher and manufacturer, because of advantages and disadvantages with both systems. We therefore hypothesize that running the RNA-to-CT™ 2-Step kit [(Applied Biosystems (AB), Foster City, CA] using a one-step protocol (as recommended) is not appropriate for quantitation of gene expression levels and should not be performed. Consequently, we ran comparative studies of the two suggested methods to evaluate their efficiency, sensitivity, and accuracy. To ensure precession, two different PCR machines were used: the StepOnePlus system and Chromo4. In addition, the RNA-to-CT™ 1-Step kit (recently launched by AB) was also used to compare its efficiency with these methods. Efficiency, sensitivity, and linearity were determined by standard curves generated using RNA isolated from C2 myoblasts to amplify the housekeeping gene GAPDH. When the RNA-to-CT™ 2-Step kit was run as a two-step reaction on the Chromo4 or StepOnePlus, respectively, not only did the efficiency increase (100±1.5% and 99.7±0.95%) but also the sensitivity (comparative threshold cycle for the lowest standard: 33.2±0.5 and 32.5±0.7) and linearity (0.997±0.001 and 0.993±0.006) compared with RNA-to-CT™ 2-Step run as one-step and RNA-to-CT™ 1-Step kit. This is the first study to demonstrate that the RNA-to-CT™ 2-Step kit is not reliable to be performed as a one-step reaction but as a two-step reaction, is even more sensitive than the newly launched RNA-to-CT™ 1-Step kit.
机译：定量RT-PCR可以一步或两步反应进行。然而，由于两种系统的优缺点，从研究者和制造商的角度出发，方法的选择引起了争议。因此，我们假设使用一步操作（建议）运行RNA-to-CT™两步试剂盒[（Applied Biosystems（AB），CA，Foster City，CA]）不适用于定量基因表达水平，应该因此，我们对这两种建议的方法进行了比较研究，以评估它们的效率，灵敏度和准确性，为确保进动，我们使用了两种不同的PCR机：StepOnePlus系统和Chromo4。还使用CT™1-Step试剂盒（AB公司最近推出的试剂盒）与这些方法进行了比较，其效率，灵敏度和线性度是通过使用从C2成肌细胞中分离的RNA扩增管家基因GAPDH生成的标准曲线确定的。 RNA-to-CT™两步试剂盒分别在Chromo4或StepOnePlus上以两步反应运行，不仅效率提高了（100±1.5％，99.7±0.95％），而且灵敏度也提高了（比较阈值）最低标准的循环：与RNA-to-CT™2步试剂盒和RNA-to-CT™1步试剂盒相比，其线性分别为33.2±0.5和32.5±0.7）和线性（0.997±0.001和0.993±0.006）。这是第一项证明RNA-to-CT™2步试剂盒作为一步反应进行但不可靠的方法，但作为两步反应，比新推出的RNA-to试剂盒更加敏感-CT™1-Step套件。

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