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一种高通量 RT-qPCR 数据筛选合适参考循环 miRNA 集的系统方法。

A systemic approach to screening high-throughput RT-qPCR data for a suitable set of reference circulating miRNAs.

机构信息

Department of Biostatistics and Translational Medicine, Medical University of Lodz, Lodz, Poland.

Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland.

出版信息

BMC Genomics. 2020 Jan 31;21(1):111. doi: 10.1186/s12864-020-6530-3.

DOI:10.1186/s12864-020-6530-3
PMID:32005151
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6995162/
Abstract

BACKGROUND

The consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. We proposed a new in silico approach of finding a suitable reference for human, circulating miRNAs and identified a new set of endogenous reference miRNA based on miRNA profiling experiments from Gene Expression Omnibus. We used 3 known normalization algorithms (NormFinder, BestKeeper, GeNorm) to calculate a new normalization score. We searched for a universal set of endogenous miRNAs and validated our findings on 2 new datasets using our approach.

RESULTS

We discovered and validated a set of 13 miRNAs (miR-222, miR-92a, miR-27a, miR-17, miR-24, miR-320a, miR-25, miR-126, miR-19b, miR-199a-3p, miR-30b, miR-30c, miR-374a) that can be used to create a reliable reference combination of 3 miRNAs. We showed that on average the mean of 3 miRNAs (p = 0.0002) and 2 miRNAs (p = 0.0031) were a better reference than single miRNA. The arithmetic means of 3 miRNAs: miR-24, miR-222 and miR-27a was shown to be the most stable combination of 3 miRNAs in validation sets.

CONCLUSIONS

No single miRNA was suitable as a universal reference in serum miRNA qPCR profiling, but it was possible to designate a set of miRNAs, which consistently contributed to most stable combinations.

摘要

背景

对于如何选择血清或血浆 miRNA 表达 qPCR 研究的参考基因，尚未达成共识，也没有一种潜在的候选基因得到令人信服的验证。我们提出了一种新的计算方法，用于寻找人类循环 miRNA 的合适参考，并基于 Gene Expression Omnibus 的 miRNA 分析实验确定了一组新的内源性参考 miRNA。我们使用了 3 种已知的归一化算法（NormFinder、BestKeeper、GeNorm）来计算新的归一化分数。我们搜索了一套通用的内源性 miRNA，并使用我们的方法在另外两个新的数据集上验证了我们的发现。

结果

我们发现并验证了一组 13 个 miRNA（miR-222、miR-92a、miR-27a、miR-17、miR-24、miR-320a、miR-25、miR-126、miR-19b、miR-199a-3p、miR-30b、miR-30c、miR-374a），可用于创建一个可靠的 3 个 miRNA 参考组合。我们表明，平均而言，3 个 miRNA 的平均值（p=0.0002）和 2 个 miRNA 的平均值（p=0.0031）比单个 miRNA 作为参考更好。在验证集，3 个 miRNA 的算术平均值：miR-24、miR-222 和 miR-27a 被证明是最稳定的 3 个 miRNA 组合。

结论

没有单个 miRNA 适合作为血清 miRNA qPCR 分析的通用参考，但可以指定一组 miRNA，它们一致地为最稳定的组合做出贡献。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/6b1b7daf8e96/12864_2020_6530_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/d55de4223110/12864_2020_6530_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/75ed37c4bcce/12864_2020_6530_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/e36bfd9af448/12864_2020_6530_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/95d47c526f38/12864_2020_6530_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/cfb7a5dc95e2/12864_2020_6530_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/56209c9edb70/12864_2020_6530_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/6b1b7daf8e96/12864_2020_6530_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/d55de4223110/12864_2020_6530_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/75ed37c4bcce/12864_2020_6530_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/e36bfd9af448/12864_2020_6530_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/95d47c526f38/12864_2020_6530_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/cfb7a5dc95e2/12864_2020_6530_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/56209c9edb70/12864_2020_6530_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f926/6995162/6b1b7daf8e96/12864_2020_6530_Fig7_HTML.jpg

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