The body Fluids’ identification by molecular methods has recently received significant research. Such is because molecular methods have an advantage over the conventional catalytic tests because of its increased confidence (5). The identification of body fluids has a great application in the forensic examination. Over the years, different approaches have been applied which are basically serological-based approaches. However, the majority of these methods are limited to being less sensitive and lacks specificity. Because of these drawbacks, there has been needing to develop a method that enables the identification of multi-fluids such as blood, menstrual blood, semen, urine, saliva and vaginal secretions among other fluids.
Recent studies demonstrate that miRNA has the potential to facilitate this identification. It has continued to gain tract is micro RNA (miRNA). MicroRNAs are short sequences of noncoding RNA molecules consisting of 18-25 nucleotides that play a crucial role in controlling physiological processes through post –transcriptional mechanisms (5). Several studies have indicated that mRNAs are usually expressed in fluids obtained during forensic studies. The findings show that MicroRNAs are better suited for the forensic study than other RNAs because of the non-specific degradation of RNAs. Such is because other RNAs are prone to the unfavorable environmental condition. However, they are stable in a dried stain foam (1). The plan of this paper is to validate the use of microRNA as a biomarker in forensic studies with through empirical evidence. The role of mRNAs is extensive and includes but not limited to biochemical mechanism regulation, augmentation of the cell and the proliferation of various cellular mechanisms. Whenever there is an alteration of pathophysiological tissues and mechanism, it causes an increase of mRNAs in human blood particularly sweat and urine. Their specificity and immovability back the application of mRNAs as candidates of biomarker circulation in the forensic examination. The miR369-3P demonstrates an example of such prowess. In a study to establish its application, using fourteen urine samples indicated that miRNA has potential to identify women fluids.
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First, the preference of MicroRNAs as an alternative biomarker in recent studies has been informed of the advantages of the latter. Unlike catalytic biomarkers, MicroRNAs are better suited to fluid identification due to their small size (3). They are also encapsulated within micro-vesicles and linked to protein complexes thus protecting them against ribonuclease degradation (3). Also, microRNA is a good biomarker due to it being easily identifiable as it is tissue specific regarding expression. Also, microRNAs are not easily destroyed by chemicals such as formalin-fixed-paraffin-embedded (FFPE) that are fundamental in a forensic examinations in which intense nucleic acid fractionation transpire (5). The microRNA can easily be extracted from the FFPE tissues gave results that show a high level of similitude to the new tissue outlines (5). All these merits are the reason for the favor that microRNAs have received in recent times.
In instances where microRNAs have been used such as the identification of various fluids in crime scenes, evidence reiterates that the best results are obtained. For instance, to cut short the conflicting aspect, four microRNAs that were evaluated in more than two studies. As well, in another previous study, 16 microRNAs expressed fluid specific microRNAs were analyzed in a set microarray data, but only 9 out of the 16 were found to have the fluid specific microRNA expression (3). For instance, semen- based microRNAs such as miRNA-135a and miRNA-135b that were early on reported were at the same time expressed in secretions from the vagina and saliva-specific microRNAs examples being miRNA-200c, miRNA-203, and miRNA-205 also were significantly articulated in vaginal secretions (1)
Still on the advantage side, when particular fluid genes are used to bring up a multiplex real time PCR assay, the gene transcripts are amplified from the body fluid as RNA isolates them by the use of the RT- qPCR (3). As well, agarose gel electrophoresis is used in their separation and detection. The results obtained confirmed body-fluid specificity by the use of RT-qPCR (3). The accuracy of isolation is usually measured by the use of RT-qPCR analysis because the other methods are not easy to detect small volumes of samples. MicroRNA truces in such scenario. For instance, Williams et al. show that when samples are analyzed using this method, the presence of microRNAs through RT-qPCR analysis are visible (5). Such is because the method of isolation is also based on the RNA yield and presence of high levels of miRs-16 and -21 causes the presence (5)
Another advantage of the microRNA is that there is a multiplex of techniques for identification of mRNA that have been advanced and deployed so far. However, the limitations of mRNA use due to their instability have led to an insignificant acceptance. Thus the study of forensic tissue specificity has been evaluated by the use of microRNAs using microarray and reverse quantitative transcription PCR (RT- qPCR) analysis. After use of microRNA in an experiment instead of mRNA all the samples that were used successfully passed the QC test (1; 5).
However, when the High throughput technique (HTS) is used to analyze the samples of body fluid through the Illumina Hi-seq using the NEBNext Multiplex small RNA library, Prep set whereby small RNAs within samples sequence reads are identifiable (4). Studies of raw sequences of samples are usually similar but the sequence reads for vasectomized semen are higher compared to those of whole semen of seminal plasma thereby can lead to false positive result (.4).
The high-throughput technique used in microRNA sequencing has challenges, for example, the presence of small RNA bacteria in body fluids causes unnecessary competition for the reagent used and leads to reverse transcription (2). Such is caused because of the direct hindrance to the percentage of sequencing reads that already had an extra insight of being human microRNAs. When tests were done on fluids that contained high bacterial load with low RNA load, it resulted into less identifiable microRNAs. Based on this, there could be a limitation of the total count of microRNAs that are to be evaluated (2). At the same time, the small sized sample found in for forensic study are of low depth thus difficult to find out some of the microRNAs that were already noted by early studies as only those collected in huge volumes were identifiable (1).
However, Williams et al. note that this shortcoming can be countered through the use of microRNAs cultured in clinical settings. As such, there is an evaluation of RNA isolates to ensure that maximum number of RNA is obtained and microRNA that can be amplified. Reverse transcription quantitative (PCR) is preferred because previously the UV spectrophotometry applied is not accurate in determining low volumes of microRNAs. RT-qPCR is used in specificity testing of samples obtained from fluid samples of the body such as saliva, blood, faeces, and semen from several individuals (1).
Reverse transcription quantitative PCR has led to the identification of microRNAs that were not expressed in the use of the high throughput sequencing. The merit of RT- qPCR is that it is more sensitive and specific thus can give accurate information of the microRNA that is under investigation (1). Also, this technique can be used in the forensic study to reduce the burden of the workload in that it is quick compared to the previous techniques that could take long before results will be realized.
The existence of an amplifiable microRNA extracts derived from DNA makes it easier to identify body fluids obtained from forensic samples. Such will effectively minimize objections usually experienced in the use of novel body fluid that requires several procedures for the final findings to be realized. The presence of a well-defined multiplex DNA extracts could easily lead to advancement in forensic analysis in a descent way (2; 5).
Human genome microRNA data has significantly increased with over 1700 detailed information of mature microRNAs. This will positively curb the challenges that were previously experienced due to the use of fewer microRNAs database (2). Also, use of a highly expressed gene will help in giving rise to more accurate and sensitive results.
Regardless of the evidence provided herein that shows the rationale behind the preference of microRNA in the identification of body fluids, the method has some drawbacks. For instance, when a mature miRNA is employed in the fluid identification, it makes it impossible for the techniques to draw a difference between the various miRNAs that develops. The reason for this is that mature miRNA results from a very long strand of primary-miRNA which often comprises of several myriads/thousands of nucleotides (5). It thus coalesces to previous miRNAs that contain between 70 and 100 nucleotides and also forms between 18 and 25 mature nucleotide strands. Such cleavage exhibited by mature miRNA (2).
As well, isolation is made difficult because of the variation of the various strands since and due to the reality that some sequence have a different nucleotide. Wang et al. remarks that the difference in nucleotide s due to modifications invoked to enhance the stability and functioning of the miRNA (5). However, this scenario can get remedy through the application of the stem-loop technique. The shortening of the miRNA that contains various nucleotides implies that the annealing temperatures were immense and made it difficult to copy long strands.
References
(1) Park, J.; Park, S.; Kwon, O.; Lee, H.; Kim, J.; Seok, H.; Lee, W.; Lee, S.; Kim, Y.; Woo, K.; Kim, S. Microarray Screening And Qrt-PCR Evaluation Of Microrna Markers For Forensic Body Fluid Identification. ELECTROPHORESIS 2014, 35 , 3062-3068.
(2) Courts, C.; Madea, B. Specific Micro-RNA Signatures for the Detection of Saliva and Blood in Forensic Body-Fluid Identification. Journal of Forensic Sciences 2011, 56 , 1464-1470.
(3) Juusola, J.; Ballantyne, J. Mrna Profiling For Body Fluid Identification By Multiplex Quantitative RT-PCR. Journal of Forensic Sciences 2007, 0 , 1-15.
(4) Seashols-Williams, S.; Lewis, C.; Calloway, C.; Peace, N.; Harrison, A.; Hayes-Nash, C.; Fleming, S.; Wu, Q.; Zehner, Z. High-Throughput Mirna Sequencing And Identification Of Biomarkers For Forensically Relevant Biological Fluids. ELECTROPHORESIS 2016, 37 , 2780-2788.
(5) Wang, Z.; Zhang, J.; Wei, W.; Zhou, D.; Luo, H.; Chen, X.; Hou, Y. Identification Of Saliva Using Microrna Biomarkers For Forensic Purpose. Journal of Forensic Sciences 2015, 60 , 702-706.
