發(fā)布時(shí)間：2019-01-13 20:43

【摘要】：【目的】采用實(shí)時(shí)熒光定量PCR技術(shù)(qRT-PCR)對(duì)基因表達(dá)進(jìn)行分析時(shí),選擇適當(dāng)?shù)膬?nèi)參基因是獲得準(zhǔn)確分析結(jié)果的關(guān)鍵。在豬睪丸分子生物學(xué)研究中,表達(dá)穩(wěn)定的蛋白編碼內(nèi)參基因和micro RNA(miRNA)內(nèi)參基因均有哪些目前尚不清楚。本研究旨在篩選出合適的內(nèi)參基因,為準(zhǔn)確定量不同時(shí)期豬睪丸組織中目的基因的表達(dá)提供可靠依據(jù)。【方法】選用8個(gè)不同發(fā)育時(shí)期(E 90、D 1、D 30、D 60、D 90、D 120、D 150、DM)的豬睪丸組織為材料,采用Trizol裂解法提取各樣品的總RNA,利用Nan Drop ND-2000分光光度計(jì)檢測(cè)總RNA的濃度和純度。設(shè)計(jì)并合成已報(bào)道的豬其他組織中表達(dá)相對(duì)穩(wěn)定的5個(gè)編碼內(nèi)參基因(GAPDH、TBP、β-actin、SDHA和B2M)以及5個(gè)miRNA內(nèi)參基因(U6、ssc-miR-17-5p、ssc-miR-26a、ssc-miR-27a和ssc-miR-103)的特異性引物序列,逆轉(zhuǎn)錄得到cDNA模板后,按1:10梯度稀釋為7個(gè)濃度梯度進(jìn)行qRT-PCR反應(yīng)以構(gòu)建標(biāo)準(zhǔn)曲線。并對(duì)10個(gè)候選內(nèi)參基因在各時(shí)期睪丸組織中的表達(dá)情況進(jìn)行實(shí)時(shí)熒光定量全面檢測(cè),采用Ge Norm法對(duì)定量結(jié)果進(jìn)行綜合分析,最后根據(jù)內(nèi)參基因穩(wěn)定性值(M值)的大小篩選出最穩(wěn)定的參考基因;M值越小,表明內(nèi)參基因的穩(wěn)定性越好,反之則越差�！窘Y(jié)果】對(duì)GAPDH、TBP、β-actin、SDHA、B2M、U6、ssc-miR-17-5p、ssc-miR-26a、ssc-miR-27a和ssc-miR-103 10個(gè)候選內(nèi)參基因的熔解曲線進(jìn)行分析發(fā)現(xiàn),均無(wú)非特異擴(kuò)增及引物二聚體,說(shuō)明各內(nèi)參基因特異性良好,qRT-PCR反應(yīng)的專一性高;以Ct值為縱坐標(biāo),相對(duì)拷貝數(shù)的對(duì)數(shù)為橫坐標(biāo)進(jìn)行標(biāo)準(zhǔn)曲線分析可知,各內(nèi)參基因在系列稀釋的濃度梯度內(nèi)具有良好的線性關(guān)系;通過(guò)對(duì)10個(gè)候選內(nèi)參基因在不同時(shí)期豬睪丸組織中的表達(dá)穩(wěn)定性分析發(fā)現(xiàn),蛋白編碼基因中,最穩(wěn)定的為TBP,最不穩(wěn)定的是GAPDH;miRNA候選內(nèi)參中,最穩(wěn)定的為U6,最不穩(wěn)定的是ssc-miR-26a。【結(jié)論】成功篩選出豬睪丸組織基因表達(dá)分析中穩(wěn)定表達(dá)的內(nèi)參基因TBP和U6,可作為豬睪丸組織基因表達(dá)研究中最佳內(nèi)參基因候選者。
[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.
【作者單位】： 湖南農(nóng)業(yè)大學(xué)動(dòng)物科學(xué)技術(shù)學(xué)院/畜禽遺傳改良湖南省重點(diǎn)實(shí)驗(yàn)室;
【基金】：國(guó)家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)資金(CARS-36)
【分類號(hào)】：S828

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