Abstract:

Real-time quantitative RT-PCR (qRT-PCR) technology, with quantitative accuracy, high sensitivity and high-throughput characteristics, has been widely used in gene expression analysis. Based on the relative quantitative analysis, qRT-PCR data must be normalized with one or more proper and stable internal reference genes. House-keeping genes are customarily used as endogenous references for relative quantification. But not a single gene can act as a universal reference reported so far. Most of the traditional housekeeping genes are unable to ensure accurate normalization in qRT-PCR. Based on the tremendous gene-chip expression data and public deposited EST data, new reference genes with superior stability were selected and verified with qRT-PCR. The research progress of reference genes in plant qRT-PCR was reviewed and aspects to be considered in future reference gene selection were also discussed.

Key words: real-time quantitative reverse transcription PCR, reference genes, geNorm, gene expression

摘要：

实时荧光定量RT-PCR(real-time quantitative reverse transcription PCR, qRT-PCR)具有定量准确、灵敏度高和高通量等特点,已被广泛应用于基因的表达分析。常规qRT-PCR采用相对定量进行分析,其关键步骤是选择合适的稳定内参基因进行校正和标准化。持家基因被广泛用作内参基因,但在所有生理条件下均稳定表达的理想内参基因并不存在。大多数传统内参基因已不能满足qRT-PCR准确定量的要求。基于基因芯片表达数据和EST数据库并结合qRT-PCR,可以筛选稳定性好的新的内参基因。简要综述了植物qRT-PCR内参基因的研究进展,并就内参基因的选择中应注意的问题进行了探讨。

关键词: 实时荧光定量RT-PCR, 内参基因, geNorm, 基因表达