小桐子低温胁迫下microRNA实时荧光定量PCR内参的筛选与比较

doi:10.13560/j.cnki.biotech.bull.1985.2019-0042

摘要/Abstract

摘要： 选择合适的内参对实时荧光定量PCR（qRT-PCR）结果的准确性极其重要,在microRNA（miRNA）的qRT-PCR分析中尤为如此。通过筛选适宜分析小桐子低温胁迫下miRNA定量表达的内参,为小桐子及其他物种miRNA的qRT-PCR分析提供有用的理论参考。基于以前的小RNA-seq结果,以低温处理的小桐子为材料,挑选11个候选内参基因,用实时荧光定量PCR技术检测它们在不同样本中的表达量,采用GeNorm、NormFinder和BestKeeper软件进行表达稳定性综合分析。结果表明,表达最稳定的基因是miR6448和U6,miR6448的Ct值为23左右,表达丰度适中;U6的Ct值为10左右,表达丰度高;因此,miR6448可作为小桐子低温胁迫下表达丰度适中的miRNA qRT-PCR的内参基因;U6可作为小桐子低温胁迫下丰度较高的miRNA qRT-PCR的内参基因。

关键词: 小桐子, 实时荧光定量PCR, 内参基因, miRNA, 低温胁迫

Abstract: Screening appropriate reference gene is of essential importance for the accuracy of quantitative real-time PCR（qRT-PCR）results,and it is especially true in qRT-PCR analysis of microRNAs（miRNAs）. Screening suitable reference genes for miRNA qRT-PCR analysis in Jatropha curcas under chilling stress may provide important reference genes for qRT-PCR analysis of miRNAs in J. curcas and other species. Eleven candidate genes were selected from the chilling-treated J. curcasbased on our previous small RNA-seq results,and their expression levels in different samples were detected by qRT-PCR. The expression stability was analyzed by GeNorm,NormFinder,and BestKeeper software. The results showed that the most stable expression were miR6448 and U6 genes. miR6448 had moderate expression abundance and its Ct was 23. U6 gene had high expression abundance,and the CT was about 10. Therefore,miR6448 can be used as the moderate expression abundance reference gene for miRNA qRT-PCR analysis in J. curcas under chilling stress. U6 gene can be used as the high abundance reference gene for miRNA qRT-PCR analysis in J. curcas under chilling stress.

Key words: Jatropha curcas, quantitative real-time PCR, reference gene, miRNA, chilling stress

孔春艳, 陈永坤, 王莎莎, 郝大海, 杨宇, 龚明. 小桐子低温胁迫下microRNA实时荧光定量PCR内参的筛选与比较[J]. 生物技术通报, 2019, 35(7): 25-32.

KONG Chun-yan, CHEN Yong-kun, WANG Sha-sha, HAO Da-hai, YANG Yu, GONG Ming. Screening and Comparison of Reference Genes for microRNA Quantitative Real-time PCR in Jatropha curcas Under Chilling Stress[J]. Biotechnology Bulletin, 2019, 35(7): 25-32.

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