Erickson Heidi S, Albert Paul S, Gillespie John W, Wallis Benjamin S, Rodriguez-Canales Jaime, Linehan W Marston, Gonzalez Sergio, Velasco Alfredo, Chuaqui Rodrigo F, Emmert-Buck Michael R
Pathogenetics Unit, Laboratory of Pathology and Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Lab Invest. 2007 Sep;87(9):951-62. doi: 10.1038/labinvest.3700659. Epub 2007 Jul 23.
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.
诸如定量逆转录酶（qRT）-PCR等基因表达测量技术需要一种标准化策略，以便在生物样本之间进行有意义的比较。通常，这是通过使用一种内源性看家基因来实现的，该基因被认为在研究的样本中表现出稳定的表达水平。对于在有限量的mRNA（如来自显微切割（MD）组织的mRNA）中能够多精确地测量特定基因，存在一些担忧。为了解决这个问题，我们评估了三种不同的方法用于显微切割样本的qRT-PCR标准化；显微切割过程中的细胞计数、总RNA测量和内源性对照基因。数据表明，细胞计数和总RNA在研究开始时校准输入量方面都很有用，但没有提供足够的精度来作为标准化标准。然而，内源性对照基因可以准确地确定目标基因相对于整个细胞转录组的相对丰度。综上所述，这些结果表明，如果采用适当的标准化策略，就可以从MD样本中进行精确的基因表达测量。
