The Screening Of Human Tissue-specific CircRNAs Molecules And Its Application In Forensic Medicine

Objective: Tissue residual marks are very representative of violent crime scenes,involving tissue fragments,knife and gun wipes or residual tissue stains on clothing and so on.Analyzing the existence and possible cell sources of biological samples related to crime is the key to reveal the nature of crime,especially in the process of violent crime scene reconstruction involving cadaver dismemberment,organ and tissue identification can provide important information supplement for the source attribution of DNA.In recent years,a new type of non-coding RNA--ring RNA?circular RNAs,circ RNAs?is gradually entering the field of vision of researchers and has become a new research hotspot in the field of RNA.The expression level of circ RNAs is tissue-specific,and circ RNAs has a closed ring structure,which is not easy to be degraded by exonuclease,which is more stable than linear RNA,and has more advantages for forensic analysis of obsolete degradation samples.In this study,human tissue-specific circ RNAs markers were screened from TSCD?Tissue-Specific circ RNA database,TSCD??http://gb.whu.edu.cn/TSCD/?database and literature data,and human tissue samples were collected,and the tissue expression specificity of candidate circ RNAs molecular markers was analyzed by q RT-PCR method,in order to find tissue-specific circ RNAs molecular markers and apply them to the identification of human organs and tissues in forensic medicine.Methods:1.Sample collection and preservation: 1 in the process of forensic autopsy,5 individual tissue samples?numbered as 1Mel No.5?were extracted for circ RNAs tissue specificity verification.There were 5 cases of heart,brain,liver,skin and skeletal muscle,1 case of lung,kidney,spleen,pancreas,thyroid,cerebellum,stomach and fat.Each sample was about 2cm,300 mg,excluding organs with obvious corruption or pathological changes.After the samples were extracted,they were immediately soaked in RNA protective solution of 20 times the volume of tissue blocks,and stored at-80 ? after overnight at 4 ?.2 the tissue samples of one autopsy individual?No.6??including heart,brain,liver,skin and skeletal muscle tissues?were extracted and used to study the stability of circ RNAs.Each sample was about 5cm3.After the tissue was extracted,the tissue was directly divided into about the size of 0.8cm3,placed in a petri dish and placed in a constant temperature and humidity box with a temperature of 15 ? and a relative humidity of 45%.RNA was extracted and analyzed on 0 days,7 days,14 days,21 days,28 days and 56 days respectively.3 A total RNA sample of human heart,cerebral cortex,liver and skeletal muscle was purchased from TAKAR Company.2.Screening of circ RNAs loci: 22 tissue-specific circ RNAs markers with high expression abundance in 5 kinds of adult tissues?heart,liver,skin,skeletal muscle,lung?were screened from TSCD database,and 3 brain tissue-specific circ RNAs markers were screened from the literature to verify tissue specificity.3.Extraction and quantification of RNA: the total RNA,of human tissue samples was extracted by TRIzol method and the quality and purity of RNA were evaluated by Nano Q micro-spectrophotometer.The RQS?RNA Quality Score?value of RNA mass fraction was measured by Labchip GX Touch RNA Assay to evaluate the integrity of RNA.4.q RT-PCR verification candidate circ RNAs:1 selected 18 s r RNA,?-actin,GAPDH,U6 as candidate internal reference genes,evaluated the expression of four internal reference genes in different tissues,and selected markers with stable expression in different tissues as internal reference genes of q RT-PCR.2 reverse?divergent?primers were designed on both sides of the cyclization site,and the expression level of tissue-specific candidate circ RNAs in different human organs and tissues was detected by SYBR Green q RT-PCR technique.Record the CT value?CT value refers to the number of PCR cycles experienced when the fluorescence signal of the fluorescence reporter group reaches the threshold in the exponential growth phase?,and use ? CT?candidate circ RNA?= CT?candidate circ RNA?-CT?internal reference gene?to represent the expression level of a circ RNAs in a certain tissue;the amplified products were agarose gel electrophoresis,and the amplification products were observed;the primer specificity and product sequence characteristics of circ RNAs were verified by Sanger sequencing.5.Latent cross tissue test: RNA samples of human lung,kidney,spleen,pancreas,thyroid,cerebellum and digestive tract were extracted and tested by q RT-PCR,and the specificity of each circ RNAs was judged according to the results of CT value and agarose gel.6.Sensitivity test: the purchased human organ tissue RNA?heart,cerebral cortex,liver,skeletal muscle?and the extracted No.3 skin tissue RNA were diluted by 10-fold series gradient dilution: the initial template was added to 100 ng,10 ng,1 ng,0.1ng and 0.01 ng,respectively.7.Evaluation of circ RNAs stability: for human organ and tissue corruption samples prepared in constant temperature and humidity box,q RT-PCR and agarose gel electrophoresis were used to analyze the expression level of circ RNAs and evaluate the stability of circ RNAs at 0 day,7 days,14 days,21 days,28 days and 56 days respectively.8.Statistical analysis: the above experimental results were analyzed by single factor analysis of variance?ANOVA?with Excel and SPSS v21.0 software.Results:1.Evaluation of candidate internal reference genes: among the four candidate internal reference genes 18 s,?-actin,GAPDH and U6,the expression of U6 in different tissues was more stable.2.In this experiment,a total of 25 circ RNAs,were screened from the database and literature,of which 12 circ RNAs could design ideal back-to-back primers.Verified by q RT-PCR method,8 circ RNAs were confirmed to have obvious tissue specificity?P<0.01?.They are chr4:95529209 | 95561601 +?heart specificity?,hsa_circ₀000296,hsa_circ₀000417?brain specificity?,chr19:10183599 | 10184111 +?liver specificity?,chr1:152324865 | 152325090-?skin specificity?,chr2:152544139 | 152550950-,chr2:152544805 | 152551143-,chr2:152496868 | 152499867-?skeletal muscle specificity?.The results of agarose gel electrophoresis showed that the amplified products of heart-specific circ RNA chr4:95529209 | 9556160 + were clear in myocardial tissue,low in brightness in liver tissue,and no obvious product band in other brain,skin and skeletal muscle tissues,suggesting that the circ RNAs could distinguish heart,liver and brain,skin and skeletal muscle tissues in an all-or-nothing way.Liver specific circ RNA chr19:10183599 | 10184111 + amplified products showed clear bands in liver tissues,low brightness bands in skeletal muscle tissues,no obvious product bands in other heart,brain and skin tissues,and no expression in these three kinds of tissues.The circ RNAs can also distinguish liver,skeletal muscle from heart,brain and skin tissues in an all-or-nothing way.3.Sanger sequencing: all the 8 sites were sequenced successfully,and the splicing sites were all predicted nodes,and the sequence was consistent with the results reported in the database and literature.4.Atent crossover tissue test: the expression levels of 8 circ RNAs in latent crossover tissues were lower than their specific expression tissues,and some circ RNAs were not expressed in some latent crossover tissues?CT>35?or the expression level was very low,showing relatively good tissue specificity.5.Sensitivity test: using the q RT-PCR method,among the 8 circ RNAs,the lowest detectable RNA addition of chr4:95529209|95561601+,chr19:10183599|10184111+ is 0.1ng,and the lowest template of the other circ RNAs is 0.01 ng.6.circ RNAs stability test: Chr4:95529209|95561601+ site could identify heart tissue in 21 days group,while other circ RNAs sites could accurately identify the corresponding target tissue in 56 days.And obvious bands could be observed in agarose gelConclusions:CircRNAs is expected to become another important molecular marker for tissue and organ recognition.In this study,25 human tissue-specific circ RNAs sites were selected from TSCD database and literature,and 8 kinds of circ RNAs molecules were successfully verified to have tissue specificity of heart?1?,brain?2?,liver?1?,skin?1?and skeletal muscle?3?.The stability of circ RNAs in human tissue samples stored for 56 days was evaluated,and the q RT-PCR method for circ RNAs quantification was constructed with high sensitivity.The experimental results show that circ RNAs has ideal stability and different expression levels in human organs and tissues,and can be used as a new genetic marker for forensic humoral identification.