犬细小病毒感染F81细胞microRNA荧光定量PCR内参基因的筛选

Screening of reference genes for microRNA fluorescence quantitative PCR in CPV-infected F81 cells

犬细小病毒(canine parvovirus, CPV)是犬急性胃肠炎的主要病原之一,对犬类的健康造成极大危害。MicroRNA(miRNA)是小的、非编码的RNA,在转录后水平调节基因表达。荧光定量PCR(RT-qPCR)是评估miRNA表达水平最常用的方法,而合适的参考基因在RT-qPCR数据归一化中起着至关重要的作用。本研究在分析F81细胞空白对照组和感染CPV组小RNA高通量测序结果基础上,结合参考文献,在CPV感染后12 h和24 h,通过poly(A)延伸RT-qPCR定量方法检测候选内参基因小RNA的表达情况,并使用GeNorm、NormFinder和BestKeeper算法评估这些候选小RNA的稳定性。结果选取了5种高通量测序中表达相对稳定的miRNAs(dno-miR-186-5p、pha-miR-152、dno-miR-374a-5p、tch-miR-26a-2-5p和oga-miR-26b)和常用作参考基因的小核RNA U6(RNU6-1)、核仁小RNA(small nucleolar RNA)SNORD95以及SNORD96a,经过扩增分析及对高通量测序中差异表达的dno-miR-7-5p的定量验证发现,这些候选小RNA均具有较好的稳定性。其中pha-miR-152在检测样品中稳定性最好,可在CPV感染F81细胞中作为RT-qPCR法定量miRNA表达及数据均一化的合适参考基因。筛选出的pha-miR-152有助于研究miRNAs在CPV感染过程中的定量表达分析,为进一步研究F81细胞中miRNA调控CPV感染复制的分子机制研究提供可靠手段,也为其他相关物种miRNA定量研究中内参基因的选择提供参考。

Canine parvovirus(CPV) is one of the main pathogenic factors of acute gastroenteritis in dogs, and it is usually difficult to cure in dogs, which has caused great harm to the health of dogs. MicroRNAs(miRNAs) are small, noncoding RNAs that regulate gene expression at the post-transcriptional level. Reverse transcription quantitative PCR(RT-qPCR) is the most used method to evaluate the expression level of miRNA, therefore, the appropriate reference genes play a crucial role in normalizing RT-qPCR data. In this study, based on the analysis of the small RNA high-throughput sequencing results of negative control group and CPV-infected group in F81 cells, combined with references, the expression of candidate miRNAs was detected by poly(A) extension RT-qPCR method at 12 h and 24 h after CPV infection, and the stability of these candidates was evaluated using GeNorm, NormFinder and BestKeeper algorithms. 5 miRNAs(dno-miR-186-5p, pha-miR-152,dno-miR-374a-5p, tch-miR-26a-2-5p and oga-miR-26b) and small nuclear RNA U6(RNU6-1), small nucleolar RNA SNORD95 and SNORD96a, which are commonly used as reference genes were selected. After amplification analysis and quantitative verification of differentially expressed dno-miR-7-5p in high-throughput sequencing, it was found that these candidate small RNAs all had good stability, among which pha-miR-152 had the best stability in the tested samples, and could be used as a suitable reference gene for quantitative miRNA expression and data normalization via RT-qPCR in CPV-infected F81 cells. This study provides valuable information for the quantitative expression analysis of miRNAs in the process of CPV infection, provides a reliable means for further research on the molecular mechanism of miRNAs regulating CPV infection and replication in F81 cells, and can also provide a reference for the selection of internal reference genes in miRNA quantitative research of other related species.