Housekeeping gene selection for quantitative real-time PCR assays in the seagrass Zostera marina subjected to heat stress
Housekeeping gene selection for quantitative real-time PCR assays in the seagrass Zostera marina subjected to heat stress
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DOI：
10.4319/lom.2006.4.367
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发表时间：
2006-11-01
影响因子：
2.7
通讯作者：
Reusch, Thorsten B. H.
中科院分区：
文献类型：
--
作者：
Ransbotyn, Vanessa;Reusch, Thorsten B. H.
随着海洋表面温度的升高，沿海生物胁迫诱导基因表达的定量化是海洋分子生态学和进化研究的重要课题。作为定量实时PCR（QPCR）检测的先决条件，我们测试了12个候选管家基因（HKG）的信使RNA丰度和质量的正常化在生态上重要的海草种大叶藻的适用性。在比较用于识别适当的HKG的几种算法时，在geNorm或Normalware中实现的更大组HKG候选者中使用相互交叉验证的两种方法产生了基本一致的结果。使用这些方法，选择3种HKG候选物，其在靶组织（叶）中单独或组合对于施加的温度胁迫处理（TATA盒结合蛋白、翻译延伸因子、真核起始因子4A）是稳定的。实例计算显示，当使用不太稳定的HKG作为标准化参考时，假设靶基因中的假基因表达变化为2倍。这些结果强调了在用靶基因进行实验之前，需要投入相当大的努力来选择适当的HKG用于QPCR数据的标准化。
In light of increasing sea surface temperatures, quantifying the expression of stress-inducible genes in coastal organisms is an important topic of marine molecular ecology and evolution. As a prerequisite for quantitative real-time PCR (QPCR) assays, we tested the suitability of 12 candidate housekeeping genes (HKGs) for normalization of messenger RNA abundance and quality in the ecologically important seagrass species Zostera marina. In comparing several algorithms used to identify appropriate HKGs, two approaches using reciprocal cross-validation among a larger group of HKG candidates implemented in geNorm or NormFinder yielded largely consistent results. Using these approaches, 3 HKG candidates were selected that are stable alone or in combination in the target tissue ( leaf) with respect to the imposed temperature stress treatments ( TATA box binding protein, translation elongation factor, eukaryotic initiation factor 4A). Example calculations revealed spurious gene expression changes of a factor of 2 in hypothetical target genes when using less stable HKGs as reference for standardization. These results highlight the need for devoting considerable effort to selecting appropriate HKGs for normalization of QPCR data before performing experiments with the target genes.