Evaluation of suitable reference genes for normalization of real-time reverse transcription PCR analysis in colon cancer.
Evaluation of suitable reference genes for normalization of real-time reverse transcription PCR analysis in colon cancer.
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DOI：
10.1186/1756-9966-29-144
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发表时间：
2010-11-08
期刊：
影响因子：
--
通讯作者：
Jacobsen MB
中科院分区：
文献类型：
--
作者：
Sørby LA;Andersen SN;Bukholm IR;Jacobsen MB
实时逆转录聚合酶链式反应(qRT-PCR)因其方法的重复性和敏感性而被广泛应用于基因表达的定量。基因表达通过归一化到一个或多个假定在给定实验中稳定表达的参考基因来量化。这项研究的目的是验证一种标准化的实验装置，以确定转移性和非转移性结肠癌中qRT-PCR正常化的参考基因。本研究利用TaqMan低密度阵列技术对18例非转移性结肠癌患者和20例有远处转移结肠癌患者的肿瘤组织和个体匹配的正常粘膜中16个常用参考基因的表达进行了定量研究。用geNorm和NormFinder对表达稳定性进行了测定和比较。根据geNorm和NormFinder的数据，HPRT1/PPIA和IPO8/PPIA两对基因分别适合于对转移性和非转移性结肠癌患者的基因表达数据进行标准化。我们提出了一种标准化的方法来寻找最合适的参考基因(S)在每个QRT-PCR实验中使用TLDA。
Real-time reverse transcription PCR (qRT-PCR) is frequently used for gene expression quantification due to its methodological reproducibility and sensitivity. The gene expression is quantified by normalization to one or more reference genes which are presumed stably expressed throughout a given experiment. The aim of this study was to validate a standardized experimental setup to identifying reference genes for normalization of qRT-PCR in the metastatic and non-metastatic colon cancer. In this study, expression of 16 commonly used reference genes was quantified in tumour tissue and individual-matched normal mucosa in 18 non-metastatic colon cancer patients and 20 colon cancer patients with distant metastases using TaqMan Low Density Array (TLDA). The expression stability was determined and compared by means of geNorm and NormFinder. Two pairs of genes, HPRT1/PPIA and IPO8/PPIA, were identified to be suitable to normalize gene expression data in metastatic and non-metastatic colon cancer patients, according to geNorm and NormFinder respectively. We propose a standardized approach of finding the most suitable reference gene(s) in every qRT-PCR experiment using TLDA.
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