猪睾丸组织定量PCR分析中内参基因的选择

发布时间：2019-01-13 20:43

【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micro RNA(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(E 90、D 1、D 30、D 60、D 90、D 120、D 150、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Nan Drop 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10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Ge Norm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。
[Abstract]:[objective] in the analysis of gene expression by real-time fluorescent quantitative PCR (qRT-PCR), the key to obtain accurate analysis results is to select appropriate internal reference genes. In the study of pig testicular molecular biology, it is not clear which of the stable protein encodes the internal reference gene and the micro RNA (miRNA) internal reference gene. The purpose of this study was to screen out suitable internal reference genes and to provide reliable basis for quantifying the expression of target genes in pig testicular tissues at different stages. [methods] eight different developmental stages (E90 D1D30D30D60D90) were selected. The total RNA, of each sample was extracted by Trizol cleavage method. The concentration and purity of total RNA were detected by Nan Drop ND-2000 spectrophotometer. Five encoding genes (GAPDH,TBP, 尾-actin,SDHA and B2m) and five miRNA genes (U6Ssc-miR-17-5pSsc-miR-26a) were designed and synthesized in other pig tissues. The specific primer sequences of ssc-miR-27a and ssc-miR-103 were obtained by reverse transcription and then diluted to 7 concentration gradients according to 1:10 gradient. QRT-PCR reaction was performed to construct the standard curve. The expression of 10 candidate internal reference genes in testicular tissues was detected by real-time fluorescence quantitative analysis, and the quantitative results were analyzed by Ge Norm method. Finally, the most stable reference gene was selected according to the stability value (M value) of the internal reference gene. The smaller the M value, the better the stability of the internal reference gene, and the worse the vice versa. [results] for GAPDH,TBP, 尾-actin,SDHA,B2M,U6,ssc-miR-17-5p,ssc-miR-26a, By analyzing the fusion curves of 10 candidate internal reference genes of ssc-miR-27a and ssc-miR-103, it was found that there was no non-specific amplification and primer dimer, which indicated that the specificity of each internal reference gene was good and the specificity of qRT-PCR reaction was high. The standard curve analysis using Ct value as the ordinate and the logarithm of the relative copy number as the horizontal coordinate shows that each internal parameter gene has a good linear relationship within the concentration gradient of the series dilution. By analyzing the expression stability of 10 candidate internal reference genes in pig testicular tissues at different stages, it was found that the most stable gene of protein coding gene was TBP, and GAPDH; was the most unstable gene. U6 was the most stable candidate reference for miRNA, and ssc-miR-26a. [conclusion] successfully screened TBP and U6, which were stably expressed in the analysis of gene expression in pig testis. It can be used as the best candidate for gene expression in pig testicular tissue.
【作者单位】：湖南农业大学动物科学技术学院/畜禽遗传改良湖南省重点实验室;
【基金】：国家现代农业产业技术体系建设专项资金(CARS-36)
【分类号】：S828

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