Ferreira Elisa N, Maschietto Mariana, Silva Sabrina D, Brentani Helena, Carraro Dirce M
Laboratory of Genomics and Molecular Biology-A.C. Camargo Hospital, Institute of Biosciences, University of São Paulo (USP), São Paulo, Brazil.
Diagn Mol Pathol. 2010 Mar;19(1):45-53. doi: 10.1097/PDM.0b013e3181ae8186.
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.
定量逆转录聚合酶链反应（qRT-PCR）是分子医学中用于基因表达分析的标准检测方法。在临床癌症研究中通常可获得的来自切开活检/针吸活检、激光显微切割肿瘤细胞及其他生物来源的样本，产生的RNA量非常少，这限制了表达分析。因此，需要一种RNA扩增程序来评估此类样本类型的基因表达水平。当以从扩增（A）RNA转化而来的cDNA作为模板时，像qRT-PCR这样的灵敏方法所产生的相对基因表达数据的可重复性和准确性尚未得到妥善解决。在本研究中，为了妥善评估这个问题，我们在2种乳腺细胞系（C5.2和HB4a）中进行了1轮线性RNA扩增，并使用从非扩增（NA）RNA和A RNA转化而来的cDNA评估了34个基因的相对表达。使用不同稀释度的cDNA，从β-肌动蛋白或甘油醛-3-磷酸脱氢酶标准化数据中获得相对基因表达，其中比较了这2种方法在表达上的变异性和倍数变化差异。我们的数据表明，即使使用质量欠佳的RNA，1轮线性RNA扩增也适合生成具有与NA样本相似置信水平的可重复且高保真的qRT-PCR相对表达数据。在单个qRT-PCR实验中使用从A RNA和NA RNA转化而来的cDNA会明显导致相对基因表达数据出现偏差。
