Establishment Of Human RNA Quantification Method And Its Application In Forensic Body Liquid Identification

OBJECTIVE: To explore the establishment of human specific RNA quantitative system.To overcome the negative failure of RNA identification of body fluid due to the false control of in the process of human initial total RNA injection amount,and to ensure the reproducibility of the experiment.METHODS: Human-specific primers and TaqMan probes were designed and synthesized based on literature search and NCBI nucleic acid database sequence,for the housekeeping gene RNA transcript(COX1),which is widely expressed in tissues and cells.The human specific RNA quantitative system was established on the platform of fluorescence quantitative PCR by using the absolute standard of standard curve.In this study,the quantitative standard system of DNA standard established by TaqMan probe method was focused on its construction and verification.The primers,probes were designed with Primer 3.0 and primer express3.0,to inspect specificity of primer compared with human and other 10 primates.The stability of DNA standards was tested using the "One Independent Test 10 times" and "Two Independent Experiments separately".On the basis of this,the quantitative system of DNA standard of SYBR Green I dye method was constructed and the quantitative system of RNA standard was designed for reference.To further analyze the efficacy of the DNA standard(TaqMan)fluorescence quantitative system,the real case investigations were simulated for the samples,using 15 cases of old blood,saliva and accelerated aging semen,screening the international common body fluid identification genetic marker gene for qRT-PCR.Analysis of the effectiveness of the COX1 system by controlling the RNA initial template sample to quantitative in three steps: 50 pg RNA(Ribogreen Quant-it)for cDNA synthesis;Quantitative COX1;COX1 was quantified and fixed copies of RNA were added for cDNA synthesis: blood samples were added to 105 copies of RNA,and 30000 copies of saliva samples RNA,semen samples were added to 1000 copies of RNA.The sensitivity,stability and repeatability of this quantitative system were analyzed by comparing the COX1 quantification.RESULTS:COX1 quantitative system in human specificity,sensitivity,stability are fully demonstrated excellent system performance.Specificity: Highly specificity is kept even in 10 primates.On the one hand,it can effectively eliminate the RNA interference of bacteria,fungi and other non-human biological in sample.on the other hand it is as a genus identification tool,more sensitive,accurate than the real test reagents,with a wide range of applicability.Sensitivity: When 50 pg RNA was uniform added,the tissue-specific gene Ct values range of three samples is larger,and fluorescence quantitative curve is no regular distribution.The Ct value of SPTB gene in blood range is from 34.53 to 42.4,and the difference was 7.87 cycles.The value range of saliva HTN3 gene is from 34.53 to 42.4 and the difference is5.6 cycles.The difference value of semen PRM2 gene was 13.370 cycles.The copy of the sample is not same using COX1 standard system quantitative: 15 copies of the blood sample copy number difference11863.243,saliva sample copy number difference 31918.901,semen sample copy number difference 387.741.The initial sample size of the three groups is adjusted to the fixed copy number,and the tissue specific genes are changed obviously.The blood sample Ct value is reduced to3.9062 cycles.33% of the negative samples are converted to positive;the salivary samples Ct values gathered between 27-28,31.5-33 cycles;semensamples Ct values reduced to 7.804.The change in Ct value above can be visually observed from the degree of fluorescence quantitative curve dispersion.The above data fully proved that sensitivity of COX1 quantitative system is very high.Stability: In the 10 independent tests for up to six months of standard,the 10 absolute quantitative standard curves had only minor changes.The slope,intercept,R2 and efficiency coefficients were respective 1.31,0.79,0.03 and 1.82.In the independent experiment,the fluorescence quantitative curve is also highly coincident.When the absolute quantitative curve is made with two sets of sample data,the data points are almost all on the fitted linear equation,R2 = 0.9953.The stability of the COX1 standard can be used to ensure repeatability of the experiment and the effectiveness of the data.CONCLUTION: Quantitative system of human specific RNA was established in this study,and it is proved that RNA quantification system is highly specific,sensitive and stable by RNA identification of body fluid system.