体液转录组分析与新型体液标志物的挖掘

doi:10.3969/j.issn.1671-2072.2025.06.006

摘要/Abstract

摘要： 目的利用转录组测序获取体液斑长链RNA转录谱，从中筛选具有体液鉴定和体液供者个体识别应用前景的RNA标志物。方法收集外周血、精液、阴道分泌物和月经血各5例样本以及9例唾液样本并提取总RNA，去除核糖体RNA以富集样本的信使RNA（messenger RNA，mRNA）、长链非编码RNA（long non-coding RNA，lncRNA）以及环状RNA（circular RNA，circRNA），构建转录组文库并测序，获得5种体液的长链RNA表达谱。随后，对5种体液样本进行体液差异表达分析，筛选出与其余体液样本均存在显著表达差异的体液特异性基因，并利用实时荧光定量PCR（real-time fluorescence quantitative PCR，qPCR）验证基因的体液特异性。结果经过对5种体液mRNA、lncRNA和circRNA表达谱的刻画，共发现了19 942个mRNA、42 206个lncRNA和151 927个circRNA，并通过差异分析识别出了4 091个体液特异性mRNA、1 052个特异性lncRNA和855个特异性circRNA。从上述候选特异性基因中筛选出目标体液中表达稳定且与其余体液表达差异显著的48个基因，利用qPCR完成其体液特异性的初步验证，其中过往未曾报道的体液特异性基因有38个，包括23个特异性mRNA、3个特异性lncRNA和12个特异性circRNA。结论本研究绘制了5种法医学常见体液的长链RNA表达谱，挖掘了大量具有体液特异性的RNA标志物，进一步完善了编码区内单核苷酸多态性（coding single nucleotide polymorphim，cSNP）的可同时用于体液鉴定和个体识别的候选基因列表。

关键词: , 法医物证学；体液鉴定；转录组测序；RNA；qPCR

Abstract: Objective The aim of this study is to obtain expression profiles of long RNA of five forensic body fluids by transcriptome sequencing, and explore prospective RNA markers for body fluid identification (BFID) and individual identification of body fluid donors. Methods The long RNAs of peripheral blood, semen, vaginal secretion and menstrual blood samples collected from five volunteers and saliva collected from nine volunteers were enriched by rRNA depletion, followed by library preparation and sequencing. Differential gene expression analysis was performed between each two of the five body fluids to identify body fluid-specific genes which show expression difference with the others. The body fluid specificity of target genes was evaluated via real-time fluorescence quantitative PCR（qPCR）. Results The expression profiles of mRNA, lncRNA and circRNA were obtained from five body fluids, and a total of 19 942 mRNAs, 42 206 lncRNAs and 151 927 circRNAs were detected. The differential gene expression analysis indicates 4 091 mRNAs, 1 052 lncRNAs and 855 circRNAs with potential body fluid specificity. A total of 48 genes were selected with stable expression in target body fluid and significant differential expression with the others, and their specificity were validated by qPCR. Among these BFID markers, 38 were novel genes, including 23 specific mRNAs, 3 specific lncRNAs and 12 specific circRNAs. Conclusion Our study measures the long RNA expression profiles in five forensic body fluids, explores a series of novel RNA markers with body fluid specificity and abundant RNA polymorphisms, and provides a candidate gene list with cSNP for body fluid identification and individual identification in forensic medicine.

Key words: forensic genetics, body fluid identification, transcriptome sequencing, RNA, qPCR

中图分类号:

DF795.2

于欢, 张更谦. 体液转录组分析与新型体液标志物的挖掘[J]. 中国司法鉴定, 2025(6): 48-59.

YU Huan, ZHANG Gengqian. The Screening of Novel Markers for Body Fluid Identification by Whole Transcriptome Sequencing[J]. Chinese Journal of Forensic Sciences, 2025(6): 48-59.

参考文献

[ 1 ] 李岩. 法医相关体液鉴定技术的研究进展[J]. 科技展望，2015，25（11）：106-107.
[ 2 ] VIRKLER K，LEDNEV I K. Analysis of body fluids for forensic purposes：From laboratory testing to non-destructive rapid confirmatory identification at a crime scene[J]. Forensic Science International，2009，188（1/2/3）：1-17.
[ 3 ] TOBE S S，WATSON N，DAÉID N N. Evaluation of six presumptive tests for blood，their specificity，sensitivity，and effect on high molecular-weight DNA[J]. Journal of Forensic Sciences，2007，52（1）：102-109.
[ 4 ] AKUTSU T，WATANABE K，SAKURADA K. Specificity，sensitivity，and operability of RSIDTM-urine for forensic identification of urine：Comparison with ELISA for Tamm-Horsfall protein[J]. Journal of Forensic Sciences，2012，57（6）：1570-1573.
[ 5 ] LIU Z D，WANG J Q，LI Z Q，et al. mRNA for body fluid and individual identification[J]. Electrophoresis，2025，46（1/2）：44-55.
[ 6 ] WOHLFAHRT D，TAN-TORRES A L，GREEN R，et al. A bacterial signature-based method for the identification of seven forensically relevant human body fluids[J]. Forensic Science International： Genetics，2023，65：102865.
[ 7 ] YUEN Z W，SHANMUGANANDAM S，STANLEY M，et al. Profiling age and body fluid DNA methylation markers using nanopore adaptive sampling[J]. Forensic Science International：Genetics，2024，71：103048.
[ 8 ] 赵一霞，胡胜，叶健，等. mRNA分析在体液斑迹组织来源推断中的应用研究进展[J]. 刑事技术，2019，44（5）：388-394.
[ 9 ] HAAS C，NEUBAUER J，SALZMANN A P，et al. Forensic transcriptome analysis using massively parallel sequencing[J]. Forensic Science International：Genetics，2021，52：102486.
[10] HAAS C，HANSON E，ANJOS M J，et al. RNA/DNA co-analysis from blood stains—results of a second collaborative EDNAP exercise[J]. Forensic Science International：Genetics，2012，6（1）：70-80.
[11] HAAS C，HANSON E，ANJOS M J，et al. RNA/DNA co-analysis from human saliva and semen stains—results of a third collaborative EDNAP exercise[J]. Forensic Science International： Genetics，2013，7（2）：230-239.
[12] HAAS C，HANSON E，ANJOS M J，et al. RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains：Results of a fourth and fifth collaborative EDNAP exercise[J]. Forensic Science International：Genetics，2014，8（1）：203-212.
[13] CAO H F，WAHLESTEDT C，KAPRANOV P. Strategies to annotate and characterize long noncoding RNAs：Advantages and pitfalls[J]. Trends in Genetics，2018，34（9）：704-721.
[14] CHUANG T D，KHORRAM O. Expression profiling of lncRNAs，miRNAs，and mRNAs and their differential expression in leiomyoma using next-generation RNA sequencing[J]. Reproductive Sciences，2018，25（2）：246-255.
[15] TANG Z L，WU Y，YANG Y L，et al. Comprehensive analysis of long non-coding RNAs highlights their spatio-temporal expression patterns and evolutional conservation in Sus scrofa[J]. Scientific Reports，2017，7：43166.
[16] YU C Y，KUO H C. The emerging roles and functions of circular RNAs and their generation[J]. Journal of Biomedical Science，2019，26（1）：29.
[17] KRISTENSEN L S，ANDERSEN M S，STAGSTED L V W，et al. The biogenesis，biology and characterization of circular RNAs[J]. Nature Reviews Genetics，2019，20（11）：675-691.
[18] LIU B N，SONG F，YANG Q R，et al. Characterization of tissue-specific biomarkers with the expression of circ-RNAs in forensically relevant body fluids[J]. International Journal of Legal Medicine，2019，133（5）：1321-1331.
[19] ZHANG Y Q，LIU B N，SHAO C C，et al. Evaluation of the inclusion of circular RNAs in mRNA profiling in forensic body fluid identification[J]. International Journal of Legal Medicine，2018，132（1）：43-52.
[20] LIU B N，YANG Q R，MENG H，et al. Development of a multiplex system for the identification of forensically relevant body fluids[J]. Forensic Science International：Genetics，2020，47：102312.
[21] WANG S Y，WANG Z，TAO R Y，et al. The potential use of Piwi-interacting RNA biomarkers in forensic body fluid identification：A proof-of-principle study[J]. Forensic Science International： Genetics，2019，39：129-135.
[22] LIU Z Y，WANG Q W，WANG N N，et al. A comprehensive characterization of small RNA profiles by massively parallel sequencing in six forensic body fluids/tissue[J]. Genes，2022，13（9）：1530.
[23] LIU Z D，WANG J Q，LI L S，et al. Identification of the body fluid donor in mixtures through target mRNA cSNP sequencing[J]. Forensic Science International：Genetics，2024，71：103066.
[24] ZHANG X Y，LI J，LIU J D，et al. Identification of the vaginal secretion donor in mixture stains using polymorphic cSNPs on mRNA biomarkers[J]. Forensic Science International： Genetics，2022，59：102703.
[25] LIU J D，CHENG X J，LIU F，et al. Identification of coding region SNPs from specific and sensitive mRNA biomarkers for the deconvolution of the semen donor in a body fluid mixture[J]. Forensic Science International：Genetics，2021，52：102483.
[26] CHEN S F，ZHOU Y Q，CHEN Y R，et al. fastp：An ultra-fast all-in-one FASTQ preprocessor[J]. Bioinformatics，2018，34（17）：i884-i890.
[27] DOBIN A，DAVIS C A，SCHLESINGER F，et al. STAR：Ultrafast universal RNA-seq aligner[J]. Bioinformatics，2013，29（1）：15-21.
[28] SHUMATE A，WONG B，PERTEA G，et al. Improved transcriptome assembly using a hybrid of long and short reads with StringTie[J]. PLoS Computational Biology，2022，18（6）：e1009730.
[29] PERTEA G，PERTEA M. GFF utilities：GffRead and GffCompare[J]. F1000Research，2020，9：304.
[30] ZHANG J Y，CHEN S，YANG J W，et al. Accurate quantification of circular RNAs identifies extensive circular isoform switching events[J]. Nature Communications，2020，11：90.
[31] JECK W R，SORRENTINO J A，WANG K，et al. Circular RNAs are abundant，conserved，and associated with ALU repeats[J]. RNA，2013，19（2）：141-157.
[32] LOVE M I，HUBER W，ANDERS S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J]. Genome Biology，2014，15（12）：550.
[33] ZUBAKOV D，HANEKAMP E，KOKSHOORN M，et al. Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples[J]. International Journal of Legal Medicine，2008，122（2）：135-142.
[34] LIU Z Y，YANG J Y，WANG N N，et al. Integrative lncRNA，circRNA，and mRNA analysis reveals expression profiles of six forensic body fluids/tissue[J]. International Journal of Legal Medicine，2024，138（3）：731-742.
[35] LINDENBERGH A，DE PAGTER M，RAMDAYAL G，et al. A multiplex （m）RNA-profiling system for the forensic identification of body fluids and contact traces[J]. Forensic Science International：Genetics，2012，6（5）：565-577.
[36] HANSON E，INGOLD S，HAAS C，et al. Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing[J]. Forensic Science International： Genetics，2018，34：206-221.
[37] HANSON E，DØRUM G，ZAMBORLIN M，et al. Targeted S5 RNA sequencing assay for the identification and direct association of common body fluids with DNA donors in mixtures[J]. International Journal of Legal Medicine，2023，137（1）：13-32.
[38] LIANG Q，SUN H Y，WU X Y，et al. Development of new mRNA markers for the identification of menstrual blood[J]. Annals of Clinical and Laboratory Science，2018，48（1）：55-62.
[39] LYNCH C，FLEMING R. One-step endpoint RT-PCR assays for confirmatory body fluid identification[J]. Forensic Science International： Genetics，2023，64：102856.

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