Wageningen Seed Laboratory, Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands.
Plant Cell Physiol. 2012 Jan;53(1):28-37. doi: 10.1093/pcp/pcr113. Epub 2011 Aug 18.
Quantifying gene expression levels is an important research tool to understand biological systems. Reverse transcription-quantitative real-time PCR (RT-qPCR) is the preferred method for targeted gene expression measurements because of its sensitivity and reproducibility. However, normalization, necessary to correct for sample input and reverse transcriptase efficiency, is a crucial step to obtain reliable RT-qPCR results. Stably expressed genes (i.e. genes whose expression is not affected by the treatment or developmental stage under study) are indispensable for accurate normalization of RT-qPCR experiments. Lack of accurate normalization could affect the results and may lead to false conclusions. Since transcriptomes of seeds are different from other plant tissues, we aimed to identify reference genes specifically for RT-qPCR analyses in seeds of two important seed model species, i.e. Arabidopsis and tomato. We mined Arabidopsis seed microarray data to identify stably expressed genes and analyzed these together with putative reference genes from other sources. In total, the expression stability of 24 putative reference genes was validated by RT-qPCR in Arabidopsis seed samples. For tomato, we lacked transcriptome data sets of seeds and therefore we tested the tomato homologs of the reference genes found for Arabidopsis seeds. In conclusion, we identified 14 Arabidopsis and nine tomato reference genes. This provides a valuable resource for accurate normalization of gene expression experiments in seed research for two important seed model species.
量化基因表达水平是理解生物系统的重要研究工具。逆转录定量实时 PCR（RT-qPCR）是用于靶向基因表达测量的首选方法，因为它具有灵敏度和重现性。然而，为了获得可靠的 RT-qPCR 结果，需要进行标准化以校正样品输入和逆转录酶效率，这是一个关键步骤。稳定表达的基因（即其表达不受研究中处理或发育阶段影响的基因）对于准确的 RT-qPCR 实验标准化是必不可少的。缺乏准确的标准化可能会影响结果并导致错误的结论。由于种子的转录组与其他植物组织不同，我们旨在鉴定专门用于两种重要种子模式物种（即拟南芥和番茄）种子 RT-qPCR 分析的参考基因。我们挖掘了拟南芥种子微阵列数据以鉴定稳定表达的基因，并与其他来源的推定参考基因一起进行了分析。总共，通过 RT-qPCR 在拟南芥种子样品中验证了 24 个推定参考基因的表达稳定性。对于番茄，我们缺乏种子的转录组数据集，因此我们测试了在拟南芥种子中发现的参考基因的番茄同源物。总之，我们鉴定了 14 个拟南芥和 9 个番茄参考基因。这为两种重要的种子模式物种的种子研究中基因表达实验的准确标准化提供了有价值的资源。
