Selection of Reference Genes for microRNA Real-time PCR Under Salt Stress in <italic style="font-style: italic">Armillaria gallica</italic>

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Selection of Reference Genes for microRNA Real-time PCR Under Salt Stress in Armillaria gallica

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- Selection of Reference Genes for microRNA Real-time PCR Under Salt Stress in Armillaria gallica
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 26, Issue 19, Pages: 35-42(2020)
- 作者机构：
  
  1.广东药科大学中药学院，广州 510006
  2.中国中医科学院中药资源中心道地药材国家重点实验室培育基地，北京 100700
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20202062
  CLC： R284.2;R289;R22;R2-031
- Received：28 December 2019，
  
  Published Online：24 July 2020，
  
  Published：05 October 2020
- 稿件说明：
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余祺瑞,华中一,周骏辉等.盐胁迫蜜环菌Real-time PCR内参microRNA的筛选[J].中国实验方剂学杂志,2020,26(19):35-42.

YU Qi-rui,HUA Zhong-yi,ZHOU Jun-hui,et al.Selection of Reference Genes for microRNA Real-time PCR Under Salt Stress in Armillaria gallica[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(19):35-42.
余祺瑞,华中一,周骏辉等.盐胁迫蜜环菌Real-time PCR内参microRNA的筛选[J].中国实验方剂学杂志,2020,26(19):35-42. DOI： 10.13422/j.cnki.syfjx.20202062.

YU Qi-rui,HUA Zhong-yi,ZHOU Jun-hui,et al.Selection of Reference Genes for microRNA Real-time PCR Under Salt Stress in Armillaria gallica[J].Chinese Journal of Experimental Traditional Medical Formulae,2020,26(19):35-42. DOI： 10.13422/j.cnki.syfjx.20202062.

摘要

目的

2

在利用实时荧光定量聚合酶链式反应（Real-time PCR）进行microRNA（miRNA）表达分析的过程中，选择合适的内参miRNA对数据标准化起到重要作用。

方法

2

该文挑选了13个蜜环菌候选miRNA对其前体进行生物信息分析，PMRD预测其前体相似序列，并用RNAfold对候选miRNA前体及其相似序列进行二级结构预测。利用Real-time PCR检测在受盐胁迫前后两种基因型蜜环菌（基因型A，基因型B）miRNA的表达量，并结合geNorm，NormFinder，BestKeeper软件分析其表达稳定性，筛选合适的内参。

结果

2

对候选miRNA的9条前体进行二级结构预测以及特征分析，证明预测的miRNA属于miR家族具有典型的茎-环结构且成熟的miRNA位于其前体的5'或是3'端；geNorm分析表明，基因型A蜜环菌可选择Novel-4*，Novel-9作为内参组合，基因型B可选择Novel-9，Novel-16作为内参组合；NormFinder分析结果显示，Novel-9在基因型A蜜环菌和基因型B蜜环菌中都有较好的稳定性；BestKeeper分析显示Novel-12*在基因型A蜜环菌中稳定性较好，Novel-2*在基因型B蜜环菌中稳定性较好。

结论

2

稳定性最优的内参miRNA为Novel-9，为进一步开展蜜环菌miRNA调控机制研究奠定了基础。

Abstract

Objiective

： In the process of microRNA expression analysis by quantitative Real-time polymerase chain reaction（Real-time PCR），the selection of miRNA plays an important role in data standardization.

Method

2

In this paper，13

Armillaria gallica

.Candidate miRNAs were selected for bioinformatics analysis of their precursors，and the PMRD was used to predict similar sequences of their precursors，and the RNAfold was used to predict the secondary structure of the candidate miRNAs and their similar sequences. Real-time PCR was used to detect miRNAs expression in two genotypes of

Armillaria gallica

（genotype A，genotype B） before and after salt stress，and geNorm，NormFinder and BestKeeper were used to analyze the stability of miRNAs expression.

Result

2

Secondary structure prediction and characterization of 9 candidate miRNA precursors showed that the miRNA predicted belonged to the miR family with typical stem-loop structure and the mature miRNAs were at the 5' or 3' end of the miRNA precursors.geNorm analysis showed that genotype A

Armillaria gallica

could select Novel-4* and Novel-9 as reference gene，genotype B could select Novel-9 and Novel-16 as its reference gene.NormFinder analysis showed that Novel-9 was stable in both genotype A and B

Armillaria gallica

.BestKeeper analysis showed that Novel-12* was stable in genotype A

Armillaria gallica

and Novel-2* was stable in genotype B

Armillaria gallica

.

Conclusion

2

miRNA Novel-9 is the best stable reference gene，which lays a foundation for further research on the regulation mechanism of miRNA in

Armillaria gallica

.

关键词

Keywords

references

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