Forensic Study Of Circular Rna For Distinguishing Between Peripheral And Menstrual Blood

Objective:Determination of the type and origin of the body fluids found at a crime scene is important for the crime scene reconstruction by building a link between sample donors and actual criminal acts.Blood stain is one of the most common body fluid stains in the scene of the crime,however,peripheral blood and menstrual blood are difficult to distinguish by eyes.It is critical for ascertaining the nature of the case to determine whether the blood is traumatic hemorrhage or famale physiological bleeding,especially in cases of sexual assaults.Circular RNA（circRNA）is a class of single stranded circular noncoding RNA without 5’end cap and 3’terminal poly（A）tail structure formed by covalent bond in the splicing of RNA.circRNAs are not easily degraded by exonuclease and are more stable than linear RNA.Studied show that circRNA is enriched in various tissues and often exhibit tissue-specific expression.In this study,we intends to screen differentially expressed circRNAs of peripheral blood and menstrual blood through the next generation sequencing（NGS）,validate candidate circRNAs with quantitative real-time polymerase chain reaction（qRT-PCR）,so as to find more ideal markers for distinguishing peripheral blood and menstrual blood in forensic practice.Methods:1 Sample collection and preservation:9 m L peripheral blood and 9 m L menstrual blood from 3 female each（19 years old,26 years old and 26 years old,respectively）were collected using EDTA anticoagulant vacuumization.After treated with TRIzol reagent（TRIzol:blood=3:1）,the blood samples were immersed in dry ice and transport to Beijing Novogene Co,LTD.20perpheral blood samples were collected with vein puncture vessel without anticoagulant from 20 female volunteers between the age of 18 and 35,as well as the menstrual blood samples from the same individual,and 50μL aliquots were spotted onto sterile cotton swabs.RNA was extracted immediately after air drying at room temperature for qRT-PCR validation experiments.Part of the blood cotton swabs were incubated at room temperature for 3 months.Urine sample was collected from three female individuals and the cotton swab was prepared in the same manner.2 RNA extraction:Total RNA was extracted using TRIzol method.Purity and quantity of RNA were assessed with Nano Q spectrophotometer.3 NGS and candidate selection:Sequencing was performed by Beijing Novogene Co,LTD with Illumina Hi Seq platform in PE150 sequencing mode.Differentially expressed circRNAs with statistically significant expression（padj<0.05）were selected as the targets of the follow-up study.4 Validation of candidate circRNAs by qRT-PCR:Based on the results of NGS,24 candidate circRNAs that differentially expressed in peripheral blood and menstrual blood were screened based on factors such as fold change value and expression levels.Divergent primers were designed and qRT-PCR assays were used to confirm the expression of selected circRNAs markers.The valueexpression in peripheral blood and menstrual blood.circRNAs which give“on-or off”signals in the two body fluids were detected by PCR amplification and agarose gel electrophoresis.Sanger sequencing was used to verify the PCR product sequence.5 Sensitivity of qRT-PCR assays:Serial dilutions（from 100 ng to 0.01 ng）of total RNA were used as an input for c DNA synthesis to evaluate the detection sensitivity of qRT-PCR assays.6 Time-wise stability of differentially expressed circRNAs:Validation of candidate circRNAs of aged samples which stored at room temperature for 1month,2 months and 3 months were performed with qRT-PCR assays.7 Statistical analysis:Statistical analysis were performed by one sample t-test,paired t-test and wilcoxon rank sum test with Excel and SPSS v21.0software.Results:1 circRNAs expression profiling:A total of 49625 circRNAs were identified in 3 pairs of peripheral blood and menstrual blood samples through NGS assay,including 36376 that were first observed in this study.509differentially expressed circRNAs in common were identified with the targets of padj<0.05.There were 160 circRNAs expressed in“on-or-off”features.Among them 23 circRNAs were detected only in peripheral blood and 137circRNAs in menstrual blood.2 The expression level of differentially expressed circRNAs inperipheral blood and menstrual blood:24 candidate circRNAs were detected by qRT-PCR.As showed in results,4 circRNAs（hsa_circ₀000212,novel_circ₀013653,novel_circ₀013655 and novel_circ₀01365）showed"on-or-off"characteristic in 17 of 20 individuals（85%）;In the other 3individuals in 3 replicates,the C_T value was undetectable at least once,and the remaining 2 C_T values were greater than or equal to 36,that is to say these circRNAs can be detected only in menstrual blood and undetectable in peripheral blood.But among them novel_circ₀013653 can also be detected in urine.12 circRNAs were statistically differencial expressed in peripheral blood and menstrual blood（P<0.05）.TheΔΔC_T value of 4 circRNAs,namely hsa_circ₀003203,hsa_circ₀004379,hsa_circ₀005616and hsa_circ₀067735,in menstrual blood were more than 3.322 than that in peripheral blood.That was to say,the expression levels of these circRNAs in menstrual blood were 10 times higher than that in peripheral blood.But only the difference of hsa_circ₀067735 was statistically significant.The other 8circRNAs had no statistical significance（P>0.05）.3 Test of urine:None of the circRNAs could be detected in urine except hsa_circ₀020093,novel_circ₀013653 and hsa_circ₀087960.4 Sanger sequencing:Except novel_circ₀010813,novel_circ₀025468and novel_circ₀036457 failed to be sequenced,the remaining 21 circRNAs were successfully sequenced.The predicted splice junctions were confirmed,which were consistent with the results of NGS.5 Sensitivity of qRT-PCR assays:Result shows that circRNA were detectable in the target body fluid using an amount as low as 0.1 ng total RNA.6 Time-wise stability of differentially expressed circRNAs:The effect of1 month,2 month and 3 month storage on the detection of circRNAs were tested by qRT-PCR.The C_T value of 3 circRNAs（hsa_circ₀005616,hsa_circ₀067735 and hsa_circ₀087960）in aged peripheral blood samples were slightly increased,whileΔC_T values remained unchanged.The C_T value of 4 circRNAs（hsa_circ₀000212,novel_circ₀013653,novel_circ₀013655and novel_circ₀013656）in aged menstrual blood samples remained unchanged,whileΔC_T values decreased.Conclusions:This study used high-throughput sequencing methods to analyze the circRNAs expression profiles in peripheral blood and menstrual blood for the first time.24 circRNAs differentially expressed in two body fluids were screened and verified.Our results highlight 4 circRNAs markers with"on-or-off"features expressed only in menstrual blood.The stability of circRNAs in peripheral blood and menstrual blood samples that were stored for three months was evaluated and found that there are differences in the degradation rate of circRNAs in two different blood samples.The qRT-PCR method for the quantification of circRNAs constructed by us has greater sensitivity.The experimental results show that circRNAs have greater stability and ideal expression level difference.which can be used as a new biomarker for forensic body fluid identification for further research.