猪组织中miR-103实时定量PCR分析时合适内参的确定

Identification of Suitable Reference for Quantitative RT-PCR Assays of miR-103 in Pig Tissues

定量实时PCR是miRNA表达检测的主要方法之一,而利用合适内参对定量实时PCR数据进行校正处理是确保该方法分析准确性的关键.为了确定猪正常组织中miR-103定量实时PCR检测分析的合适内参,首先采用geNorm算法和扩增效率试验对miR-196、U6 snRNA和总RNA3个候选内参进行了评价;然后以miR-103的绝对定量结果为对照,比较分析了3个候选内参的校正准确性.结果表明,miR-196的表达稳定性和扩增效率均优于U6 snRNA和总RNA;以miR-196为内参的miR-103相对定量结果同绝对定量结果具有较高的一致性,两者均显示miR-103在猪大脑中高丰度表达,在胃、小脑、小肠、心脏、肝脏和胰脏中适度表达,在肺、脾脏和腿肌中轻度表达.这些结果说明,miR-196可作为猪正常组织中miR-103定量实时PCR相对定量分析的一个合适内参.

Quantitative real-time PCR（qRT-PCR） is one of the common methods for determining the miRNA transcriptions.The proper normalization of the qRT-PCR results using appropriate references is critical for the accuracy of this method.In order to identify an optimal reference for miR-103 qRT-PCR assays in normal pig tissues,three candidate references,miR-196,U6 snRNA and total RNA,were assessed by qRT-PCR with the geNorm algorithm and the amplification efficiency of experimental validation.The accuracies of the three candidate references were compared based on the absolute quantification of miR-103 with the control.The results showed that miR-196 outperformed than U6 snRNA and total RNA both in the stability and the amplification efficiency.The relative quantization normalized to miR-196 was highly correlated to the miR-103 absolute amount.We found that miR-103 was most abundantly in the cerebrum,moderately transcribed in the stomach,cerebellum,small intestine,heart,liver and pancreas,and weak in the lung,spleen and crura muscles.These results indicated that miR-196 was a suitable reference for assaying relative levels of miR-103 by qRT-PCR in normal pig tissues.