Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples
Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples
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DOI：
10.1038/labinvest.3700659
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发表时间：
2007-09-01
影响因子：
5
通讯作者：
Emmert-Buck, Michael R.
中科院分区：
文献类型：
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作者：
Erickson, Heidi S.;Albert, Paul S.;Emmert-Buck, Michael R.
基因表达测量技术，如定量逆转录酶（qRT）-PCR需要一个标准化策略，以允许有意义的比较，在生物样品。通常，这是通过使用内源管家基因来实现的，所述内源管家基因被认为在研究的样品中显示稳定的表达水平。人们关心的是如何精确地特定的基因可以在有限数量的mRNA，如那些从显微解剖（MD）组织测量。为了解决这个问题，我们评估了三种不同的方法，用于解剖样品的qRT-PCR标准化;显微切割期间的细胞计数，总RNA测量和内源性对照基因。数据表明，细胞计数和总RNA都可用于在研究开始时校准输入量，但不能提供足够的精度作为归一化标准。然而，内源性对照基因可以准确地确定靶基因相对于整个细胞转录组的相对丰度。总之，这些结果表明，如果采用适当的归一化策略，可以从MD样品进行精确的基因表达测量。
Gene expression measurement techniques such as quantitative reverse transcriptase (qRT)-PCR require a normalization strategy to allow meaningful comparisons across biological samples. Typically, this is accomplished through the use of an endogenous housekeeping gene that is presumed to show stable expression levels in the samples under study. There is concern regarding how precisely specific genes can be measured in limited amounts of mRNA such as those from microdissected (MD) tissues. To address this issue, we evaluated three different approaches for qRT-PCR normalization of dissected samples; cell count during microdissection, total RNA measurement, and endogenous control genes. The data indicate that both cell count and total RNA are useful in calibrating input amounts at the outset of a study, but do not provide enough precision to serve as normalization standards. However, endogenous control genes can accurately determine the relative abundance of a target gene relative to the entire cellular transcriptome. Taken together, these results suggest that precise gene expression measurements can be made from MD samples if the appropriate normalization strategy is employed.