山羊肌内前体脂肪细胞诱导分化过程中内参基因的表达稳定性分析

The Expression Stability Analysis of Reference Genes in the Process of Goat Intramuscular Preadipocytes Differentiation in Goat

旨在通过比较9个内参基因在山羊肌内前体脂肪细胞诱导分化不同时期的表达水平进而最终确定适合山羊肌内前体脂肪细胞分化调控研究最佳的内参基因。本研究利用胶原酶消化法获得山羊肌内前体脂肪细胞,并以分别诱导0、1、2、3、5和6d的细胞为研究对象,利用荧光定量PCR(Real-time quantitative PCR,qPCR)技术和geNorm、NormFinder和BestKeeper程序来检测和分析9个候选内参基因(GAPDH、PPIA、18S rRNA、PPIB、UXT、RPLP0、ACTB、EIF3K和TBP)表达的稳定性,并以KLF3和KLF12的实际表达水平进行校正分析。geNorm和NormFinder程序分析表明,UXT稳定性相对最好;而BestKeeper程序分析发现,PPIB表达相对最平稳;3种软件分析结果均表明,同时使用UXT和PPIB可以准确校正在山羊肌内前体脂肪细胞诱导分化不同时期目标基因的表达;通过对KLF3和KLF12表达量的分析发现,选用筛选结果中最稳定(UXT和PPIB、UXT)和最不稳定(EIF3K和18S rRNA)的内参基因进行归一化分析结果有差异。在山羊肌内前体脂肪细胞诱导分化过程中,UXT基因是适合于山羊肌内前体脂肪细胞的成脂诱导分化研究最稳定有效的内参基因,同时使用UXT和PPIB作为内参基因能够获得更加精确的目标基因表达结果,这为后续山羊前体脂肪细胞分化调控相关基因的功能研究奠定了基础。

The aim of this study was to determine the most suitable reference genes in different periods of goat intramuscular preadipocytes differentiation in goat by comparing the expression levels of 9 candidate reference genes.The intramuscular preadipocytes of goat were obtained by collagenase digestion technique.The adipocytes induced for 0,1,2,3,5 and 6 dwere used in this study.The expression levels of the 9 candidate reference genes（GAPDH,PPIA,18 S rRNA,PPIB,UXT,RPLP〈0,ACTB,EIF3 Kand TBP）were detected by real-time quantitative PCR（qPCR）,and the expression stability was evaluated by geNorm,NormFinder and BestKeeperprograms,individually.Meanwhile,the suitability of the selected reference genes for normalization was further assessed by the expression of KLF3 and KLF12.The results showed that UXT was identified as the most suitable reference gene by both geNorm and NormFinder analysis,while the PPIB was identified as the most suitable reference gene by BestKeeper analysis.The combined use of UXT and PPIBas the reference genes resulted in the optimal selection for normalization of analyzed genes in different periods of intramuscular preadipocytes differentiation in goat based on the analysis of geNorm,NormFinder and BestKeeper;Furthermore,the expression levels of KLF3 and KLF12 normalized by the most stable reference genes（UXTand PPIB,UXT）were significantly different from that normalized by the least stable ones（EIF3 K,18 S rRNA）.In conclusion,the expression of UXT was the most stable and it was the most suitable reference genes during the differentiation of goat intramuscular preadipocyte in goat,meanwhile the combined use of UXTand PPIB was found to lead to a more accurate result for target genes normalization.These results provided a foundation for functional research of genes related to preadipocytes differentiation in goats.