Dini Pouya, Loux Shavahn C, Scoggin Kirsten E, Esteller-Vico Alejandro, Squires Edward L, Troedsson Mats H T, Daels Peter, Ball Barry A
Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
Department of Reproduction, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Mol Biotechnol. 2018 Jan;60(1):62-73. doi: 10.1007/s12033-017-0047-2.
MicroRNAs (miRNAs) have important posttranscriptional regulatory abilities, and there is considerable interest in evaluating their expression patterns in different pathophysiological states. The most common method of quantifying miRNA expression is quantitative reverse transcription PCR; however, the identification of tissue-specific and species-specific reference miRNA is a prerequisite for miRNA expression analysis. Currently, no reference genes have been described for evaluating miRNA expression in equine serum and chorioallantoic membrane (CAM) during pregnancy. The aim of the present study was to characterize reference genes for normalization of miRNA expression in CAM and serum in the pregnant equine. To identify the most stable miRNAs in serum, expression of potential candidates was evaluated in serum samples from diestrous mares, pregnant mares and geldings. To identify the most stable miRNAs in CAM, expression of potential candidates was evaluated in CAM, collected from mares at 4, 6 and 10 months of pregnancy and immediately postpartum. From a previously generated miRNA sequencing dataset, two separate lists of potential reference miRNAs were identified (serum and CAM) using the NormFinder program, in addition to the commonly used small RNA normalizers, 5S rRNA and U6 snRNA. The putative reference miRNAs were selected using geNorm and NormFinder. In case of a nonsignificant correlation between the results of ranking and stability value between these two programs, ranking from BestKeeper was also included. NormFinder and geNorm consistently identified eca-miR-21-5p, eca-let-7a-5p and eca-miR-10a-5p as the three most stable reference genes for the normalization of serum miRNAs. Within CAM samples, the average ranking obtained from the ranking of NormFinder, geNorm and BestKeeper identified eca-miR-8908a-1-5p, eca-miR-369-5p and eca-miR-106a-5p as the three most stable miRNAs. These observations provide information about equine-specific reference genes that can be used for normalizing miRNAs expression patterns in CAM and serum during the equine pregnancy.
微小RNA（miRNA）具有重要的转录后调控能力，人们对评估其在不同病理生理状态下的表达模式有着浓厚兴趣。定量miRNA表达最常用的方法是定量逆转录PCR；然而，鉴定组织特异性和物种特异性的参考miRNA是miRNA表达分析的先决条件。目前，尚无用于评估妊娠期间马血清和绒毛尿囊膜（CAM）中miRNA表达的参考基因。本研究的目的是鉴定用于标准化妊娠马CAM和血清中miRNA表达的参考基因。为了鉴定血清中最稳定的miRNA，在发情间期母马、妊娠母马和去势公马的血清样本中评估了潜在候选miRNA的表达。为了鉴定CAM中最稳定的miRNA，在妊娠4、6和10个月以及产后立即从母马收集的CAM中评估了潜在候选miRNA的表达。除了常用的小RNA标准化物5S rRNA和U6 snRNA外，利用NormFinder程序从先前生成的miRNA测序数据集中鉴定出两个独立的潜在参考miRNA列表（血清和CAM）。使用geNorm和NormFinder选择推定的参考miRNA。如果这两个程序的排名结果与稳定性值之间无显著相关性，则也纳入BestKeeper的排名。NormFinder和geNorm一致将eca-miR-21-5p、eca-let-7a-5p和eca-miR-10a-5p鉴定为标准化血清miRNA的三个最稳定参考基因。在CAM样本中，从NormFinder、geNorm和BestKeeper的排名中获得的平均排名将eca-miR-8908a-1-5p、eca-miR-369-5p和eca-miR-106a-5p鉴定为三个最稳定的miRNA。这些观察结果提供了有关马特异性参考基因的信息，可用于标准化马妊娠期间CAM和血清中miRNA的表达模式。
