High-throughput miRNA sequencing and identification of biomarkers for forensically relevant biological fluids

microRNAs (miRNAs) are small noncoding RNAs that regulate cellular processes through modulation of proteins at the translational level. They tend to be highly stable as compared to other RNA species due to their small size and protection by protein and/or lipid matrices. Thus, it is likely that miRN...

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Published inElectrophoresis Vol. 37; no. 21; pp. 2780 - 2788
Main Authors Seashols-Williams, Sarah, Lewis, Carolyn, Calloway, Chelsea, Peace, Nerissa, Harrison, Ariana, Hayes-Nash, Christina, Fleming, Samantha, Wu, Qianni, Zehner, Zendra E.
Format Journal Article
LanguageEnglish
Published Germany Blackwell Publishing Ltd 01.10.2016
Subjects
Bacteria
Blood
Body fluid identification
Body fluids
Body Fluids - chemistry
Female
Fluids
Forensic Genetics - methods
Forensic science
Genetic Markers - genetics
High throughput sequencing
High-Throughput Nucleotide Sequencing - methods
Humans
Male
microRNA
MicroRNAs - analysis
MicroRNAs - genetics
Proteins
Ribonucleic acids
Saliva
Sequence Analysis, RNA - methods
Sequencing
Serology
microRNA
Serology
Forensic science
High throughput sequencing
Body fluid identification
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Summary:microRNAs (miRNAs) are small noncoding RNAs that regulate cellular processes through modulation of proteins at the translational level. They tend to be highly stable as compared to other RNA species due to their small size and protection by protein and/or lipid matrices. Thus, it is likely that miRNAs, when fully evaluated, will make excellent candidates for body fluid identification. miRNA analysis of body fluids has been the subject of some recent interest in the forensic community. In this study, small RNAs were isolated from individual donations of eight forensically relevant biological fluids (blood, semen, vaginal fluid, menstrual blood, saliva, urine, feces, and perspiration) and subjected to next generation sequencing using the Illumina Hi‐Seq platform. Sequencing reads were aligned and annotated against miRbase release 21, resulting in a list of miRNAs and their relative expression levels for each sample analyzed. Body fluids with high bacterial loads (vaginal fluid, saliva, and feces) yielded relatively low annotated miRNA counts, likely due to oversaturation of small RNAs from the endogenous bacteria. Both body fluid specific (miRs‐200b, 1246, 320c, 10b‐5p, 26b, and 891a) and potential normalization miRNAs (let‐7g and i) were identified for further analysis as potential body fluid identification tools for each body fluid.
Bibliography:U.S. Department of Justice
ark:/67375/WNG-LSTMDRH2-6
ArticleID:ELPS5970
istex:971D0BF6CEBB4E018954D27CF40A430BB4ABDDC4
National Institute of Justice
Supplemental Table 1: High-throughput sequencing samples - donor ages, ethnicities, and gender Supplemental Figure 1: Evaluation of miR-16 abundance for the top three isolation methods for each body fluid shows no major differences in efficiency. RT-qPCR analysis of miR-16 relative abundance in order to assess isolation efficiency. Each sample was analyzed in triplicate and the average Cq value was calculated. Top: Blood, semen, vaginal secretions and menstrual secretions. Bottom: Urine, saliva, and perspiration. Supplemental Figure 2: RNA isolation from fecal samples requires a feces-specific RNA isolation method. A (Top): Total RNA yields among five original methods tested were similar, but were not consistently detectable with RT-qPCR (>75% amplification failures). The PowerMicroBiome™ Standard method produced comparable yields of total RNA with B (Bottom): successful RT-qPCR analysis of miR-16 relative abundance. Each sample was analyzed in triplicate and average Cq value calculat Supplemental Table 2: Optimal RNA isolation methods for each biological fluid, used in this study Supplemental Figure 3: Depth of annotated miRNA coverage. Sequencing reads were aligned to mirBase (v20) and total read counts of annotated miRNAs (with read count >20) calculated. Data is the average of samples sequenced for each biological fluid type, and organized in quartiles to indicate relative abundances. Top labels indicate total number of annotated sequencing reads Supplemental Table 3. Average relative expression (RPM) of candidate endogenous reference miRNAs. Supplemental Table 4: Narrowed field of body fluid specific candidate miRNAs Supporting Information
See the article online to view Fig. 1 in colour.
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content type line 23
ISSN:0173-0835
1522-2683
DOI:10.1002/elps.201600258