Barragán M, Martínez A, Llonch S, Pujol A, Vernaeve V, Vassena R
Clinica EUGIN, Barcelona, Spain.
Fundació EUGIN, Barcelona, Spain.
Andrology. 2015 Jul;3(4):797-804. doi: 10.1111/andr.12053. Epub 2015 Jun 20.
Although the male gamete participates in a significant proportion of infertility cases, there are currently no proven molecular markers of sperm quality. The search for significant gene expression markers is partially hindered by the lack of a recognized set of reference genes (RGs) to normalize reverse transcription quantitative PCR (RT-qPCR) data across studies. The aim of this study is to define a set of RGs in assisted reproduction patients undergoing different sample collection and RNA isolation methods. Twenty-two normozoospermic men were included in the study. From each man, semen was either cryopreserved by slow freezing or analyzed fresh, and, for each, RNA was extracted with either phenol-free or phenol-based methods. In two cases, both methods were used to isolate RNA. Twenty putative RGs were analyzed and their mRNA abundance across samples was estimated by RT-qPCR. To determine the genes whose steady-state mRNA abundance remains unchanged, three different algorithms (geNorm, BestKeeper and NormFinder) were applied to the qPCR data. We found that RGs such as GAPDH or ACTB, useful in other biological contexts, cannot be used as reference for human spermatozoa. It is possible to compare gene expression from fresh and cryopreserved sperm samples using the same isolation method, while the mRNA abundance of expressed genes becomes different depending on the RNA isolation technique employed. In our conditions, the most appropriate RGs for RT-qPCR analysis were RPLP1, RPL13A, and RPLP2. Published discrepancies in gene expression studies in human spermatozoa may be due in part to inappropriate RGs selection, suggesting a possible different interpretation of PCR data in several reports, which were normalized using unstable RGs.
尽管男性配子在相当比例的不育病例中起作用，但目前尚无经证实的精子质量分子标志物。寻找重要的基因表达标志物部分受到缺乏一套公认的参考基因（RGs）的阻碍，这些参考基因用于在各项研究中对逆转录定量PCR（RT-qPCR）数据进行标准化。本研究的目的是在接受不同样本采集和RNA分离方法的辅助生殖患者中确定一组参考基因。22名正常精子男性被纳入研究。从每名男性采集的精液，要么通过慢速冷冻保存，要么新鲜分析，并且对于每份精液，RNA提取采用无酚法或基于酚的方法。在两个病例中，两种方法都用于分离RNA。分析了20个假定的参考基因，并通过RT-qPCR估计它们在各样本中的mRNA丰度。为了确定稳态mRNA丰度保持不变的基因，将三种不同算法（geNorm、BestKeeper和NormFinder）应用于qPCR数据。我们发现，在其他生物学背景下有用的GAPDH或ACTB等参考基因不能用作人类精子的参考。使用相同的分离方法可以比较新鲜和冷冻保存精子样本的基因表达，而表达基因的mRNA丰度会因所采用的RNA分离技术不同而有所差异。在我们的条件下，RT-qPCR分析最合适的参考基因是RPLP1、RPL13A和RPLP2。人类精子基因表达研究中已发表的差异可能部分归因于参考基因选择不当，这表明在一些报告中对PCR数据可能有不同的解释，这些报告使用不稳定的参考基因进行了标准化。
