Screening and evaluation of endogenous reference genes for miRNA expression analysis in forensic body fluid samples

MicroRNA (miRNA)-based methods for body fluid identification are promising tools in the practice of forensic science. The selection of appropriate endogenous reference genes as normalizers for the relative quantification of miRNA expression levels using quantitative reverse transcription-polymerase...

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Veröffentlicht in:Forensic science international : genetics 2023-03, Vol.63, p.102827-102827, Article 102827
Hauptverfasser: Wei, Sunxiang, Hu, Sheng, Han, Na, Wang, Guoli, Chen, Huixiang, Yao, Qianwei, Zhao, Yixia, Ye, Jian, Ji, Anquan, Sun, Qifan
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container_title Forensic science international : genetics
container_volume 63
creator Wei, Sunxiang
Hu, Sheng
Han, Na
Wang, Guoli
Chen, Huixiang
Yao, Qianwei
Zhao, Yixia
Ye, Jian
Ji, Anquan
Sun, Qifan
description MicroRNA (miRNA)-based methods for body fluid identification are promising tools in the practice of forensic science. The selection of appropriate endogenous reference genes as normalizers for the relative quantification of miRNA expression levels using quantitative reverse transcription-polymerase chain reaction (RTqPCR) is essential to avoid errors and improve the comparability of miRNA expression level data among different body fluids. In this study, small RNAs were isolated from individual donations of five forensically relevant body fluids (peripheral blood, menstrual blood, saliva, semen and vaginal secretions). Thirty-seven samples were subjected to high-throughput miRNA sequencing. By combining our results with those obtained through a literature investigation, 28 candidate RNAs were identified. Following RTqPCR validation, the candidate RNAs were preliminarily evaluated in 15 samples to exclude miRNAs with low expression and high variation. Then, the expression levels of 10 relatively stable candidate reference RNAs in 100 samples were determined and further analysed using four commonly employed programs (geNorm, NormFinder, BestKeeper and ΔCq). According to the comprehensive stability rankings of the four algorithms, miR-320a-3p was validated as the most stable endogenous reference gene among the five forensically relevant body fluids, followed by miR-484, SNORD43, miR-320c and RNU6b. Moreover, the combined application of miR-320a-3p with RNU6b could increase the normalization effect. In addition, a total of 56 mock samples placed outdoors and indoors for different times were prepared to further evaluate the stability of candidate reference RNAs, and miR-320a-3p remained the preferred reference gene. Furthermore, the relative expression levels of publicly accepted body fluid-specific miRNAs were determined in 30 samples to verify the practicality and effectiveness of the reference genes. Our results revealed a set of alternative reference genes and could promote the development and application of miRNA-based body fluid identification by determining optional reference genes for strict normalization. •High-throughput sequencing was conducted to systematically screen reference genes for miRNA-based body fluid identification.•MiR-320a-3p identified by high-throughput sequencing was ultimately determined to be a reliable reference gene.•A total of 238 samples were tested to verify the applicability of candidate reference genes.•Mock degraded samples w
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The selection of appropriate endogenous reference genes as normalizers for the relative quantification of miRNA expression levels using quantitative reverse transcription-polymerase chain reaction (RTqPCR) is essential to avoid errors and improve the comparability of miRNA expression level data among different body fluids. In this study, small RNAs were isolated from individual donations of five forensically relevant body fluids (peripheral blood, menstrual blood, saliva, semen and vaginal secretions). Thirty-seven samples were subjected to high-throughput miRNA sequencing. By combining our results with those obtained through a literature investigation, 28 candidate RNAs were identified. Following RTqPCR validation, the candidate RNAs were preliminarily evaluated in 15 samples to exclude miRNAs with low expression and high variation. Then, the expression levels of 10 relatively stable candidate reference RNAs in 100 samples were determined and further analysed using four commonly employed programs (geNorm, NormFinder, BestKeeper and ΔCq). According to the comprehensive stability rankings of the four algorithms, miR-320a-3p was validated as the most stable endogenous reference gene among the five forensically relevant body fluids, followed by miR-484, SNORD43, miR-320c and RNU6b. Moreover, the combined application of miR-320a-3p with RNU6b could increase the normalization effect. In addition, a total of 56 mock samples placed outdoors and indoors for different times were prepared to further evaluate the stability of candidate reference RNAs, and miR-320a-3p remained the preferred reference gene. Furthermore, the relative expression levels of publicly accepted body fluid-specific miRNAs were determined in 30 samples to verify the practicality and effectiveness of the reference genes. Our results revealed a set of alternative reference genes and could promote the development and application of miRNA-based body fluid identification by determining optional reference genes for strict normalization. •High-throughput sequencing was conducted to systematically screen reference genes for miRNA-based body fluid identification.•MiR-320a-3p identified by high-throughput sequencing was ultimately determined to be a reliable reference gene.•A total of 238 samples were tested to verify the applicability of candidate reference genes.•Mock degraded samples were used for the first time to assess the usability of candidate reference RNAs.•Body fluid-specific miRNAs were analysed and discussed using different reference RNAs.</description><identifier>ISSN: 1872-4973</identifier><identifier>EISSN: 1878-0326</identifier><identifier>DOI: 10.1016/j.fsigen.2023.102827</identifier><identifier>PMID: 36642061</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Body fluid identification ; Body Fluids - chemistry ; Endogenous reference gene ; Female ; Forensic Medicine ; Forensic science ; Gene Expression Profiling ; High-throughput sequencing ; Humans ; MicroRNA ; MicroRNAs - metabolism ; Normalization ; Real-Time Polymerase Chain Reaction ; RT-qPCR ; Saliva - chemistry ; Semen - chemistry</subject><ispartof>Forensic science international : genetics, 2023-03, Vol.63, p.102827-102827, Article 102827</ispartof><rights>2023 Elsevier B.V.</rights><rights>Copyright © 2023 Elsevier B.V. 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The selection of appropriate endogenous reference genes as normalizers for the relative quantification of miRNA expression levels using quantitative reverse transcription-polymerase chain reaction (RTqPCR) is essential to avoid errors and improve the comparability of miRNA expression level data among different body fluids. In this study, small RNAs were isolated from individual donations of five forensically relevant body fluids (peripheral blood, menstrual blood, saliva, semen and vaginal secretions). Thirty-seven samples were subjected to high-throughput miRNA sequencing. By combining our results with those obtained through a literature investigation, 28 candidate RNAs were identified. Following RTqPCR validation, the candidate RNAs were preliminarily evaluated in 15 samples to exclude miRNAs with low expression and high variation. 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subjects Body fluid identification
Body Fluids - chemistry
Endogenous reference gene
Female
Forensic Medicine
Forensic science
Gene Expression Profiling
High-throughput sequencing
Humans
MicroRNA
MicroRNAs - metabolism
Normalization
Real-Time Polymerase Chain Reaction
RT-qPCR
Saliva - chemistry
Semen - chemistry
title Screening and evaluation of endogenous reference genes for miRNA expression analysis in forensic body fluid samples
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