猪睾丸组织定量PCR分析中内参基因的选择

摘要

[目的]采用实时荧光定量PCR技术(qRT-PCR)对基因表达进行分析时,选择适当的内参基因是获得准确分析结果的关键.在猪睾丸分子生物学研究中,表达稳定的蛋白编码内参基因和microRNA (miRNA)内参基因均有哪些目前尚不清楚.本研究旨在筛选出合适的内参基因,为准确定量不同时期猪睾丸组织中目的基因的表达提供可靠依据.[方法]选用8个不同发育时期(E90、D1、D30、D60、D90、D120、D150、DM)的猪睾丸组织为材料,采用Trizol裂解法提取各样品的总RNA,利用NanDrop ND-2000分光光度计检测总RNA的浓度和纯度.设计并合成已报道的猪其他组织中表达相对稳定的5个编码内参基因(GAPDH、TBP、β-actin、SDHA和B2M)以及5个miRNA内参基因(U6、ssc-miR-17-5p、ssc-miR-26a、ssc-miR-27a和ssc-miR-103)的特异性引物序列,逆转录得到cDNA模板后,按1∶10梯度稀释为7个浓度梯度进行qRT-PCR反应以构建标准曲线.并对1 0个候选内参基因在各时期睾丸组织中的表达情况进行实时荧光定量全面检测,采用GeNorm法对定量结果进行综合分析,最后根据内参基因稳定性值(M值)的大小筛选出最稳定的参考基因;M值越小,表明内参基因的稳定性越好,反之则越差.[结果]对GAPDH、TBP、β-actin、SDHA、B2M、U6、ssc-miR-17-5p、ssc-miR-26a、ssc-miR-27a和ssc-miR-103 10个候选内参基因的熔解曲线进行分析发现,均无非特异扩增及引物二聚体,说明各内参基因特异性良好,qRT-PCR反应的专一性高;以Ct值为纵坐标,相对拷贝数的对数为横坐标进行标准曲线分析可知,各内参基因在系列稀释的浓度梯度内具有良好的线性关系;通过对10个候选内参基因在不同时期猪睾丸组织中的表达稳定性分析发现,蛋白编码基因中,最稳定的为TBP,最不稳定的是GAPDH;miRNA候选内参中,最稳定的为U6,最不稳定的是ssc-miR-26a.[结论]成功筛选出猪睾丸组织基因表达分析中稳定表达的内参基因TBP和U6,可作为猪睾丸组织基因表达研究中最佳内参基因候选者.%[Objective] Reference gene is the key to obtain the accurate analysis results when apply quantitative RT-PCR (qRT-PCR) technique to identify the gene expression level.However,reference gene and microRNA (miRNA) which are used in researching the molecular biology of porcine testis are not well characterized.Screening suitable reference gene will provide a reliable evidence for the quantitative analysis of target gene in different periods of porcine testis tissues.[Method] Porcine testis tissues at 8 different developmental stages (E 90,D 1,D 30,D 60,D 90,D 120,D 150,and DM) were selected as materials in this study.Trizol method was used to extract the total RNA and the concentration and purity of total RNA were detected by ND-2000 NanDrop spectrophotometer.The primers of 5 commonly used protein coding reference genes (GAPDH,TBP,β-actin,SDHA and B2M) and 5 miRNA reference genes (U6,ssc-miR-17-5p,ssc-miR-26a,ssc-miR-27a and ssc-miR-103) were designed and synthesized to reverse transcription.cDNA templates were diluted to 7 concentration gradients with 1∶10 serial dilution to construct standard curves.The expression of 10 candidate reference genes in porcine testis tissues was systematically analyzed at specified times,then the GeNorm 3.5 was used to analyze the results.The most stable reference genes were picked up according to the stability value which called M value,the smaller the value of M,the better the stability of the reference gene;otherwise,the stability would be worse.[Result] The melting curves of 10 candidate reference genes (GAPDH,TBP,β-actin,SDHA,B2M,U6,ssc-miR-17-5p,ssc-miR-26a,ssc-miR-27a,and ssc-miR-103) indicated that the expression of all the candidate reference genes was of some specificity,no primer-dimers and nonspecific amplification.The relationship between Ct value and the logarithm of relative copy number was coincidence with linear relation according to the standard curves which were made with Ct value as ordinate and the logarithm of relative copy number as abscissas in a series of diluted concentration gradients.Analysis showed that the most stable reference gene of protein encoding gene was TBP and the most unstable was GAPDH.U6 was the most suitable gene for miRNA expression analysis and ssc-miR-26a was the most unsuitable.[Conclusion] This study has screened the most suitable reference genes (TBP and U6) to identify porcine testis gene expression level successfully.The highly ranked reference genes identified from this study can provide a theoretical basis for the selection of the most suitable reference gene to detect differences in expression rates of genes and miRNAs in porcine testis tissues.