Comparisons Of Peripheral Blood RNA Pretreatment Methods And Evaluations Of RNA Markers’ Application Value In Forensic Age Estimation

Objective:1.To explore the influence of different pretreatment methods on the quality of RNA extracted from peripheral blood samples and to choose better pretreatment methods.Based on the better pretreatment methods,we further explored whether the RNA quality were affected by the storage time,and evaluates the value of pretreatment methods in forensic applications.2.To construct the database of m RNA and mi RNA markers which are highly conservative and have significant correlation with age among different tissues.Methods:1.By consulting the domestic and foreign literatures,in addition to the most commonly used direct freezing method,the methods of storaging in TRIzol ^TM,RNAlater ^TM,PAXgene blood RNA tubeand DNA/RNA Shield ^TMwere selected,too.Finally,eight pretreatment methods were designed,such as peripheral blood with DNA/RNA shield ^TM.The differences in yield,purity and integrity of extracted RNA were analyzed in statistic level.2.The peripheral blood samples were pretreated with DNA/RNA Shield ^TM.The time gradients of cryopreservation were set as 0,5,10,15,30 and 60 days.The differences in yield,purity and integrity of extracted RNA were statistically analyzed.3.After screening m RNA and mi RNA markers that are significantly related to age in peripheral blood and brain tissue,the selected markers among different tissues were compared and analyzed,so as to screen out the markers that are consistent with age in different tissues,and builded such a database of these markers.Results:1.RNA was successfully extracted by eight pretreatment methods.The purity of RNA extracted from peripheral blood with DNA/RNA shield^TMwas 1.837±0.020,the yield was3.035±0.298μg/m L,and the RIN was 8.467±0.038.The purity of RNA extracted from peripheral blood with TRIzol^TMwas 1.893±0.030,the yield was 1.977±0.085μg/m L,and the RIN was 8.167±0.195.The purity of RNA extracted from peripheral blood with RNAlater^TMwas 1.863±0.150,the yield was 2.251±0.172μg/m L,and the RIN was 8.033±0.158.The purity of RNA extracted from blood cells with DNA/RNA Shield^TMwas 1.900±0.011,the yield was 3.847±0.170μg/m L,and the RIN was 8.200±0.129.The purity of RNA extracted from leukocyte with TRIzol^TMwas 1.814±0.016,the yield was 2.468±0.060μg/m L,and the RIN was 8.200±0.120.The purity of RNA extracted from leukocyte with RNAlater^TMwas 1.621±0.046,the yield was 2.468±0.060μg/m L,and the RIN was 8.133±0.135.The purity of RNA extracted from driectly frozen peripheral blood was 1.125±0.044,the yield was 1.728±0.287μg/m L,and the RIN was 5.583±0.132.The purity of RNA extracted from peripheral blood kept in PAXgene tube was 2.030±0.002,the yield was 2.457±0.123μg/m L,and the RIN was 9.433±0.154.By paired t-test,the quality of RNA obtained by the eight pretreatment methods were statistically analyzed.In terms of purity,leukocyte with RNAlater^TMand driectly frozen peripheral blood performed worst,while the other six methods performed well and had no statistical difference.In terms of yield,blood cells with DNA/RNA Shield^TMperformed best.Comparing with the other five methods,the yields of RNA extracted from peripheral blood with TRIzol^TM,leukocyte with RNAlater^TMand driectly frozen peripheral blood were lower,and the driectly frozen peripheral blood method showed statistically difference with the other five methods.In terms of integrity,peripheral blood kept in PAXgene tube method performed best,directly frozen peripheral blood method performed worst,and the other six methods were statistically different from the methods of extracted from driectly frozen peripheral blood,except for the two methods of DNA/RNA shield^TM.2.The purity of RNA stored in six time gradients ranged from 1.815 to 1.952.With the increase of storage time,RNA yield decreased from 4.516 to 1.039,and RNA integrity decreased from 8.533 to 7.150.The single factor analysis of variance（ANOVA）showed that the RNA yield and integrity decreased with the increase of storage time,too.3.After the analysis and processing of age-related data,2194 age-related m RNA markers and319 age-related mi RNA markers were selected in peripheral blood,556 age-related m RNA markers and 127 age-related mi RNA markers were selected in brain tissue.Through the comparative analysis among different tissues,a highly conserved marker database was constructed containing 320 m RNA markers and 28 mi RNA markers.Conclusions:1.After analyzing the differences in purity,yield,and integrity of RNA obtained by the eight pretreatment methods in this study,the pretreatment method of adding DNA/RNA shield^TMto peripheral blood is recommended as the pretreatment method of samples in field sampling.2.The storage time of RNA extracted by treating peripheral blood with DNA/RNA shield^TMmethod can get to 60 days if stored at low temperature,and the extracted RNA can meet the requirements of downstream experiments such as high-throughput sequencing.3.Through comparative analysis of different tissues,a total of 320 m RNA and 28 mi RNA markers were screened and the database was constructed.These RNA markers are highly correlated with age and highly conserved among tissues,reflecting the potential application value of RNA in age estimation of forensic science.