Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance.
Reference genes for accurate gene expression analyses across different tissues, developmental stages and genotypes in rice for drought tolerance.
复制标题
DOI：
10.1186/s12284-016-0104-7
复制
发表时间：
2016-12
期刊：
影响因子：
--
通讯作者：
Kohli A
中科院分区：
文献类型：
--
作者：
Pabuayon IM;Yamamoto N;Trinidad JL;Longkumer T;Raorane ML;Kohli A
定量逆转录聚合酶链式反应(qRT-PCR)是定量检测基因表达水平的常规方法。这项技术通过与被分析样本中统一表达的内部对照基因进行比较来确定目标基因的表达。结果的重复性和可靠性在很大程度上取决于所使用的参考基因。为了在水稻抗旱性研究中实现成功的基因表达分析，参考基因的选择应该基于不同变量表达的一致性。我们的目标是提供在水稻不同组织、不同发育阶段和不同基因型之间保持一致的参考基因，从而提高qRT-PCR分析的数据质量。从4个普遍表达的基因家族中筛选出10个候选参考基因，通过分析公共微阵列数据集，包括水稻的多个器官、发育阶段和水分利用状况。这些基因通过qRT-PCR实验进行评估，并进行严格的统计分析，以确定最佳参考基因。一个泛素异源基因作为单一参考基因表现出最好的基因表达稳定性，而另一个泛素和两个亲环素异基因的3个基因组合是最好的参考基因组合。比较qRT-PCR和内部根微阵列数据，证明了所识别的参考基因用于监测干旱相关候选基因差异表达的可靠性。从常用的基因家族中确定了特定的同源基因，用于基于qRT-PCR的基因表达分析，用于水稻耐旱性研究。这些指标在治疗、基因、组织和生长阶段的变量中都是稳定的。基于可用的资源，建议进行单基因和/或三基因集分析。本文的在线版本(doi：10.1186/s12284-0160104-7)包含补充材料，授权用户可以使用。
Quantitative reverse transcription PCR (qRT-PCR) has been routinely used to quantify gene expression level. This technique determines the expression of a target gene by comparison to an internal control gene uniformly expressed among the samples analyzed. The reproducibility and reliability of the results depend heavily on the reference genes used. To achieve successful gene expression analyses for drought tolerance studies in rice, reference gene selection should be based on consistency in expression across variables. We aimed to provide reference genes that would be consistent across different tissues, developmental stages and genotypes of rice and hence improve the quality of data in qRT-PCR analysis. Ten candidate reference genes were screened from four ubiquitously expressed gene families by analyzing public microarray data sets that included profiles of multiple organs, developmental stages, and water availability status in rice. These genes were evaluated through qRT-PCR experiments with a rigorous statistical analysis to determine the best reference genes. A ubiquitin isogene showed the best gene expression stability as a single reference gene, while a 3-gene combination of another ubiquitin and two cyclophilin isogenes was the best reference gene combination. Comparison between the qRT-PCR and in-house microarray data on roots demonstrated reliability of the identified reference genes to monitor the differential expression of drought-related candidate genes. Specific isogenes from among the regularly used gene families were identified for use in qRT-PCR-based analyses for gene expression in studies on drought tolerance in rice. These were stable across variables of treatment, genotype, tissue and growth stage. A single gene and/or a three gene set analysis is recommended, based on the resources available. The online version of this article (doi:10.1186/s12284-016-0104-7) contains supplementary material, which is available to authorized users.