Preliminary Screening And Verification Of Skeletal Muscle CircRNA In Forensic Death Time Inference

Objective Accurate inference of postmortem interval?PMI?has always been the focus and difficulty in forensic science,but so far no unified detection methods and indicators have been obtained.In this study,we constructed circ RNA expression profiles in skeletal muscles,and screened circ RNAs that were associated with PMI,furthermore,the selected circ RNAs were validated,and the correlation between their relative expression and death time was discussed.This will provide a new idea and indicator for the inference of death time in forensic science.Methods?1?Chip experiment:?1?Two individuals with external temperature and humidity that are relatively consistent and whose death time is within 24 hours were selected,and taking the third and fourth intercostal muscle tissue on the right side and place it in PBS buffer to simulate the natural degradation process in an artificial climate chamber with a temperature of 18 °C and a humidity of 50 %;?2?Taking 50 g tissue on 0 days,3 days,5 days,7 days,10 days respectively,and freezing them in liquid nitrogen,and then total RNA was extracted.;?3?The extracted RNA was subjected to quality inspection,and after passing the quality test,experiments such as reverse transcription,probe labeling,and gene chip detection were performed to finally obtain a circ RNA expression profile.?2?Screening and verification of circ RNAs:?1?The intercostal muscle samples of 18 dead individuals that were subsequently collected and consistent with the previous external temperature and humidity were placed in an artificial climate chamber of the same conditions;?2?An appropriate amount of tissue was taken at 1-10 days after death for RNA extraction and reverse transcription,and the resulting c DNA was placed in a-80 °C refrigerator for subsequent experiments.?3?According to Norm Finder software,the most stable internal reference genes among GAPDH,?-Actin and mir133a-3p were screened;?4?Circ RNAs with significant differences from day 0 of death were screened based on the resulting circ RNA expression profiles,and circ RNAs with too large or too small molecular weight and low expression levels were removed;?5?The selected circ RNAs are searched in the literature for their verified primer sequences,and if there is the same circ RNA,the sequences are directly used,and if no relevant literature reports,the circbase is used to find the gene sequence,and the primer-blast is used for primer design;?6?The selected circ RNAs were subjected to Q-PCR experiments using the c DNA obtained above to verify their correlation between relative expression and PMI in 18 samples.?3?Statistical analysis: The data were statistically analyzed using SPSS 13.0 software.Results A total of 13156 circ RNAs were detected in skeletal muscle tissue.Compared with the relative expression of circ RNAs at 0 days of death,there were 353 circ RNAs with a significant downward trend on the third day,259 on the 5th day,and 273 on the 7th day,and 290 on the 10 th day,in addition,all of them were P < 0.05,Fold change >2.But the overall downward trend of circ RNAs within 10 days are only 7 such as hsa_circ₀000284,hsa_circ₀001136,hsa_circ₀001727,hsa_circ₀005251,hsa_circ₀001871,hsa_circ₀001946,hsa_circ₀001971 and so on.According to the Norm Finder software,the mir133a-3p was found to be the most stable in this experiment.The selected 7 circ RNAs were verified in 18 samples,and only 5 circ RNAs such as hsa_circ₀000284,hsa_circ₀001136,hsa_circ₀001727,hsa_circ₀005251,hsa_circ₀001871,etc.,with the increase of death time,the relative expression decreased significantly and statistically significant.?P < 0.05?.The expression levels of two circ RNAs,hsa_circ₀001946 and hsa_circ₀001971,were negative.Conclusion?1?Muscle tissue can be used as a candidate for determining the time of late death.?2?Mir133a-3p is the most stable internal reference gene in this experiment.?3?These circ RNAs were subsequently verified and five genes such as hsa_circ₀000284,hsa_circ₀001136,hsa_circ₀001727,hsa_circ₀005251,hsa_circ₀001871 decreased significantly with the increase of death time.?4?circ RNA can be used as a relatively stable biomarker for the study of death time inference.