结肠癌实时逆转录聚合酶链反应分析中合适内参基因的评估。
Evaluation of suitable reference genes for normalization of real-time reverse transcription PCR analysis in colon cancer.
机构信息
Quality and Research Department, Ostfold Hospital Trust, 1603 Fredrikstad, Norway.
出版信息
J Exp Clin Cancer Res. 2010 Nov 8;29(1):144. doi: 10.1186/1756-9966-29-144.
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSION
We propose a standardized approach of finding the most suitable reference gene(s) in every qRT-PCR experiment using TLDA.
背景
实时逆转录聚合酶链反应（qRT-PCR）由于其方法学的可重复性和敏感性，常被用于基因表达定量。通过将基因表达标准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进行定量。本研究的目的是验证一种标准化的实验方案，以确定用于转移性和非转移性结肠癌 qRT-PCR 标准化的参考基因。
方法
在这项研究中，使用 TaqMan 低密度阵列（TLDA）在 18 例非转移性结肠癌患者和 20 例远处转移的结肠癌患者的肿瘤组织和个体匹配的正常黏膜中定量了 16 个常用参考基因的表达。通过 geNorm 和 NormFinder 确定并比较了表达稳定性。
结果
根据 geNorm 和 NormFinder，分别有两对基因，HPRT1/PPIA 和 IPO8/PPIA，被确定为适合于转移性和非转移性结肠癌患者的基因表达数据标准化。
结论
我们提出了一种使用 TLDA 为每个 qRT-PCR 实验找到最适合的参考基因的标准化方法。
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