| Background Different kinds of body fluid stains are often encountered in forensic field investigations,such as bloodstains,semen spots,saliva spots,sweat spots,etc.Bloodstains are the most common biological test materials in crime scenes and one of the most important items in forensic physical evidence inspection.When blood leaves the human body,it will be affected by many factors,such as physical,chemical and biological factors.The process of pollution,degradation and corruption is very rapid.And the longer it leaves the body,the greater the change of blood composition.Especially the destruction of blood macromolecule protein and DNA will increase the difficulty of individual recognition.Therefore,the corruption of bloodstain seriously affects its test results.The test process and difficulty of bloodstains is higher than that of clinical blood test.How to accurately and efficiently identify the body fluid stains left in the crime scene is the key problem that forensic workers must solve in practical work.As a kind of single strand endogenous non coding RNA molecule(composed of about 22 nucleotides),mi RNA has high tissue-specific and stage specific expression,and is not easy to degrade(strong stability).These advantages provide the basis for the application of mi RNA in forensic work,and have important significance and good application prospects in forensic physical evidence detection(such as body fluid,tissue identification,etc).At the same time,as a small RNA,mi RNA has strong stability,but in theory,it will inevitably be affected by RNase.In the past,most of the clinical studies on mi RNA stability were preserved under freezing conditions(-20? or-80?,liquid).In recent years,some researchers made body fluid spots from blood,semen,etc.and stored them at room temperature to explore the stability of mi RNA.In the practical work of forensic medicine,most biological samples have a small amount,and often exist indoors or outdoors(natural conditions),sometimes even destroyed by human.In addition,in the aspect of forensic physical evidence,the long-term preservation of biological samples is one of its important work,the samples are mostly stored at room temperature.Therefore,it is particularly important to investigate the stability of mi RNA in samples under different environmental conditions,and then determine the optimal storage conditions for the samples.Objective To explore the influence of different experimental conditions(temperature,humidity,UV light intensity,outdoor etc.)on the stability of mi RNA in bloodstains,and provide the basis for the preservation conditions of samples involved in mi RNA detection in forensic cases,meanwhile,by analyzing whether U6 can exist stably under a variety of experimental conditions,we can determine whether it is suitable as a reference gene in the blood trace test.Methods The bloodstains were stored in different temperature(-20?,4?,25?,37?),relative humidity(30%±0.5%,45%±0.5%,60%±0.5%,75%±0.5%),ultraviolet light intensity(25uw/cm3,44uw/cm3),natural conditions(rain,no rain),laboratory conditions(RT,low humidity and dark).When samples were stored to a series of estimated test times,reverse transcription-polymerase chain reaction(RT-PCR)and real time quantitative PCR(RT-q PCR)were performed to detect the existence of blood-specific mi RNA markers(mi R-16-5p,mi R-451a)and one reference gene(U6 sn RNA)in bloodstained samples.Then the data were analyzed by means of variance analysis,fitting and regression analysis,etc.And the stability and degradation characteristics of mi RNAs and U6 in bloodstains were observed.Results and discussion In this study,a series of experimental conditions were established to explore the stability of mi RNAs and U6 in bloodstains,and to provide reference for the storage conditions of samples in forensic physical evidence inspection.On the one hand,by studying the stability of mi RNAs(mi R-16-5p,mi R-451a)in bloodstains,it is found that 37? are more suitable for short-term storage(within one week),while-20? is more suitable for long-term storage(15-180 days).When bloodstains were placed in different relative humidity conditions,the degradation rate of mi RNAs was closely related to environmental humidity,with higher humidity(relative humidity 75%)would accelerate the degradation rate of mi RNAs.In the study of different UV intensity conditions,the higher UV intensity could lead to the lower degradation rate at early stages(within one week),while there was no significant difference in the degradation rate at later stage(P > 0.05).Through the study of different natural conditions(rain or no rain),it was found that rain had an adverse effect on the stability of mi RNAs.Furthermore,the target mi RNAs could be successfully detected in bloodstain samples stored for 3 and 6 years under laboratory conditions(RT,low humidity and darkness),and the contents of mi R-16-5p and mi R-451a significantly decreased in 6 years compared with those stored for 3 years(P = 0.044,P = 0.004).On the other hand,according to the study on the stability of U6 in bloodstains,it was found that high temperature and high humidity would accelerate the degradation rate of U6 in bloodstains,and with the extension of time,the content of U6 in bloodstains would gradually decrease.And in the study of different UV intensities,the higher UV intensity could lead to the lower degradation rate at early stages(within one week),while in the later stage,there was no significant difference(P > 0.05),it was similar to the stability of mi RNAs.Then in the different natural conditions(with rain or without rain),rain could accelerate the degradation rate of U6.Furthermore,U6 could be successfully detected in bloodstains stored for 3 and 6 years under laboratory conditions,and there was no significant difference in U6 content between samples at different times(P = 0.913).Conclusion In this study,the suitable storage conditions for bloodstains were selected by exploring the influence of different environmental factors on mi RNAs and U6 in bloodstains.Our results showed that mi R-16-5p and mi R-451 a in bloodstain samples had strong stability under various experimental conditions and period,which may be due to their short length and not easy to degrade.Meanwhile,different kinds of mi RNAs showed different degradation characteristics.Compared to mi R-16-5p,mi R-451 a showed higher abundance and stability,which may be related to the sequence number of GC or CG contained in mi RNA.However,high humidity and rain water have a strong destructive effect on bloodstain samples,which makes the content of mi RNAs and U6 decrease sharply.On the other hands,in the previous studies,U6 was thought to have strong stability and could be used as a reference gene in the relative quantification of mi RNAs for the identification of body fluid sources.However,in this study,we found that high temperature and high humidity would accelerate the degradation of U6 in bloodstain samples,and with the extension of storage time,the content of U6 would gradually decrease.Our results showed that different mi RNAs in bloodstains had different degradation rates,but they all show strong stability,and they have been successfully detected in all experimental samples,indicating that mi RNA can be used as a stable biomarker in bloodstains,which has good forensic application value.Moreover,this study is expected to provide a reference for the storage of forensic physical evidence.First of all,to ensure the full sterilization of the carrier(glass plate,cotton swab,blood collection card,etc.)before collect blood will be conducive to its subsequent storage.In addition,37? is more suitable for the short-term storage of bloodstain samples,and if long-term storage is needed,-20?,low humidity can ensure the success rate of sample detection. |
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