miRNA as Biomarkers in Forensic Body Fluids Identification

miribonucleic acid as Biomarkers in Forensic Body Fluids Identificationmiribonucleic acid profiling What does non consort for product line and peeing identificationSarah S. Silva a, b, Teixeira, A.L b, MJ Carneiro de Sousa a,c and Medeiros, R.a, ba ICBAS, Abel Salazar Biomedical Sciences Institute, University of Porto, 4050-313 Porto, Portugalb molecular Oncology group, Portuguese Institute of Oncology, 4200-072 Porto, Portugalc _ National Institute of Legal Medicine and Forensic Sciences, North Branch, 4050-167 Porto, Portugal thieveIn rhetoricals, the identification of line of merchandise, semen or vaginal secretions drop represent an important support for a criminal investigation. They can be engagementd as a ancestor of deoxyribonucleic acid but similarly can hold, just by their presence, the most probative value. Through the years umpteen methodologies were utilise to discern them but altogether presented serious drawback. Lately, mRNA surged as a likely tool for be facile identification but their sensibility were a serious disadvantage, even more pronounced in forensic samples. Since 2009, miRNA profiling surged as a possible tool as a substantiating test in forensics repayable to their tissue particular pattern of materialization. Unlike mRNAs they are much more stable imputable to their proprieties whose makes them little prone to degradation processes.In this report, we studied the lookingal patterns of miR-127, miR-221 and RNU-48 in 50 samples of urine and declivity in regularise to define whether or not they could be used as biomarkers for urine or blood identification.Even though our aim was to assess whether or not our miRNAs could be considered as biomarkers, we came across 2 others intimacying conclusions the impact of RNA laurels in miRNAs quantification and which miRNA cannot be used as a normalisation gene for blood and urine identification.Key words miRNA profiling, Forensic, Serology, ashes fluids, biologi cal biomarkers1- IntroductionHuman soundbox fluids are important components to rely on a criminal investigation 1, 2. As a press of fact, a complainants body fluids present on items belonging to a suspect or vice versa holds the most probative value. For example, in a exercise of a sexual assault in a child, where a DNA profile rec e preciseplaceed from the child bedding and underwear coincide with his experience DNA profile, can we consider his father responsible for the sexual assault? In a case like this, it is not enough to recover a DNA profile but it is also crying to acknowledge its source. If no serological test were done, in court, the presence of DNA could be explained as a result of the presence of epithelial cells in the child clothing which is totally common when it comes from a sibling. On the other hand, if serological tests linked the DNA profile to semen it would be way more tall(prenominal) to explain its presence there.Beyond the probative value that body fluid may have in a crime scene, it is also important to acknowledge them to optimize protocols to conduct a reliable DNA profiling 3, 4. For example, DNA extraction processes are different for blood and urine. If we conducted the protocol of blood extraction in urine samples it may result in a reduced quality of the extracted DNA e transform any conclusive DNA profile 3, 4. There is why, body fluids identification is considered as crucial measuring stick in criminal investigation.For some, it seems easy to make out body fluids such as blood ( colour in), urine (smell) or even sperm (texture) however, when dried, washed or mixed with other components their identification may not be that easy 1. It is important to highlight that in court, there is no such thing as It seems to be sperm because it looked like it and have the same particular texture, it is needed an undeniable proof that it is sperm. serologic test are used in forensic biology to allow the sensing and identificati on of body fluids in both native form or as a residue left at a crime scene. Serological tests are divided in two study fields likely and confirmatory test. Presumptive tests rely on methodologies that are sensitive and performed quickly, yet they are not unique(predicate) to the body fluid. Those tests can only indicate if the fluids might be present and do not unambiguously adduces its presence. On the other hand, confirmatory tests are indeed specific to the body fluid we seek to identify. As presumptive tests, confirmatory testing is sensitive however, it takes a lot more time.Idealistically, we should have a barrage fire of confirmatory test for all important body fluids in dress to reliably detect and identify them. Unfortunately, there is a large cluster of presumptive tests and far less of confirmatory ones. Moreover, till designation no confirmatory test is able to reliably differentiate blood from menstrual blood which is an definitely important body fluid in sex ual cases.Over the last years, mRNA profiling became a target for body fluid identification due to its tissue specific patterns. Still, mRNA susceptibility to degradation by physical or chemical factors was an unquestionable drawback. In order to sidetrack this problem, miRNA surge with a real potential as a confirmatory test. MiRNAs are small non-coding RNAs with more or less than 22 nucleotides of length that, combined with the RNA-induced silencing complex, seems to regulate a major part of human gene (5 e 6 do meu artigo). Moreover, their tight relationship with Argonaute proteins, they are much less susceptive to both biotic and abiotic factors. In 2009, Hanson and colleagues were the first to introduce miRNA profiling and soon enough others followed. Those studies pointed out a large collection of miRNAs with potential as biomarker, however very few were confirmed by more than one group which revealed the lack of reproducibility of results. Moreover, when some tried to replic ate the results of others, they failed.For this report, we choose to test four miRNAs in both blood and urine of 50 healthy individual and study their demeanour within those body fluids.2- Material and methodsWe conducted an expression profiling of 50 healthy individuals. The case group was composed by Caucasian individuals with no major pathological condition in order to erase a variable that could alter miRNAs profiles. Peripheral venous blood (Xml) and urine were collected from each subject following the obtainment of a written sensible consent from all subjects.After collected the samples were processed. The samples were used for miRNAs extraction with GRS microRNA Kit (Grisp) according to the manufacturers instructions. Subsequently, miRNa priorly extracted were used as a template for cDNA synthesis using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). To quantify miRNA expression, real-time PCR assays were performed with a StepOne System using TaqManUnivers al Master Mix II (Applied Biosystems). The target miRNAs were amplified by a set of designed primers for miR-127-5p, miR-221*, miR-222* and RNU48. miR-222* was used as a normalization gene miRNAs relative quantifications.The data synopsis was performed using the StepOne Software v2.2 (Applied Biosystems).Statistical analysis was carried out by the computer software IBMSPSSStatistics (Version 22.0). In order to assess any statistical alte rations in our normalized miRNAs expression we used 2Ct method and Students t test.3- Results3.1- Cycle threshold vs RNA purityUrine samples were processed and the resulting pellet was diluted in 1ml of Tripure. visually a wide range of pink colour was noticeable within our urine samples. Those with a darksome pink were related with samples with a more substantial pellet unlike those with a less gigantic pellet who presented themselves with a lighter colour. After miRNA extraction, we quantify miRNA expression of miR-222 in urine samples and perc eived that only few of them were detected. Interestingly, only the ones with a lighter colour were indeed detected. This tricky situation could be explained by the ratio of absorbance at 260 nm and 280 nm which is used to assess the purity of RNA. In this case, lighter colour was also an indicator of a greater ratio, on the other hand, those with higher optical density had a very low ratio, far from the ratio of 2.0 which is generally accepted as pure for RNA. In order to sidetrack this delicate situation, we choose a sample (MU26) that has an optimal 260/280nm ratio and diluted the other samples to score their optical density with Tripure. Posteriorly, we choose 5 samples to test and noticed a long decrease of Ct in the samples processed with the optimized protocol (Fig.1). The deviation of Ct value is very material, nearly 6 Ct, demonstrating that RNA purity is clearly a factor that challenge miRNA profiling. As showed, miRNA quantification goes with a low concentration or can go totally undetected when 260/280nm ratio is low however, when optimized, miRNA concentration increased significantly. As give tongue to previously, different reports indicated miRNAs as biomarkers for human body fluids identification though, when others tried to replicate them, they failed. Our results shows that for the same sample, different degrees of purity can decide whether or not a miRNA is detected, once it definitely affect their concentration. There is why, RNA purity needed to be optimal otherwise it may lead to unreliable results, which could explain, the failed attempts done by some authors when trying to replicate others results.Figure 1 Cycle threshold vs RNA purity. This figure presents the Ct values of miR-222 taken from 5 samples processed with both normal and optimized protocol (first and second column respectively). It is showed that the considerable fall of Ct values correlates with an increase of 260/280nm ratio.3.2 Normalization geneIn qRT-PCR, data norm alization is imperatively required for quantification analysis 5-7. The integration of an invariant endogenous essay, also called as acknowledgment gene, has as its chief(prenominal) objective correct systematic technical and/or experimental errors 6, 8.For this essay, we choose to use RNU-48 as our university extension gene for the data normalization. Widely used as normalization gene, RNU-48 is expected to have a stable pattern among samples. However, within our essay the opposite transpired. As showed in figure 2, RNU-48 was the one with a major standard deviation when compared with other 3 miRNAs analyzed which make it inappropriate as an endogenous go for for our essay.Seemingly, we were not the only ones that reason out this, Sapre and colleagues also assumed that RNI-48 was inadequate as an endogenous conceal due to its systematic perturbation in its expression 9.Remarkably, the unexpected miR-222 profile remained barely unaffected and presented no significant differen ce between urine and blood. miRNA-222 behaviour within our samples was surprising once, it is being aimed for its deregulation by many other groups. Here, it does not present any variation within samples, any variation among both body fluids, it did even remained stable within different stages of age and do not alter with gender. This particular behaviour is expected of endogenous controls. Therefore, we decided to use miR-222 as our reference gene in order to normalise our data.3.3 miRNAs as biomarkersSince 2009, miRNAs has been a target for forensic researcher, especially in forensic serology. The importance of both detection and identification for body fluids in criminal investigation is undeniable. Scientifically speaking, 5 years is such a short time to develop reliable new methodologies and, as already lay out by some authors, there is still so much to do.Here, we choose 4 miRNAs and decided to study their expression level in urine and blood samples. As stated earlier, we cho ose miR-222 as our endogenous control for our data normalisation due to its behaviour within our samples. As showed in figure 4, we can state that all miRNAs considered have different expressional patterns and all of them probabilistically significant (PRNU-48 is the one with a major difference between urine and blood. The one used numerous times as an endogenous control is upregulated near 141 times more in blood than in urine supporting our decision to not use it to normalize our data.Till now, a minor number of miRNAs have been acknowledged as tissue specific at least reliably. By definition, miRNAs are considered tissue specific when theyre found with high abundance in a specific tissue while it has low or non-existent expression in others. That differential profile patterns would allow body fluids reliable identification and serve as a significant confirmatory test. Considering our results, we can conclude that miR-127, miR-221 and RNU-48 are not suitable for neither blood nor urine identification. Despite a significant difference of expression, they do not present the expected expressional patterns to be considered as a full biomarker.Table 1 miRNA detection in both urine and blood samples and its corresponding fold exchange within the body fluids.As we stated within our introduction, the miRNAs considered as biomarkers for body fluid identification in other reports have been difficult to replicate. We believe that those difficulties are linked to several(prenominal) factors as environmental factors, methodologies, age, gender, pathologies among several others. We know that miRNAs expression levels do alter with both biotic and abiotic factors, there is why we try to play down the impact of those within our samples excluding, as example, acute pathological conditions. Despite considering that miR-127, miR-221 and RNU-48 are unsuitable for urine and blood identification, we wanted to study their expressional behavior within samples with different stages of age and gender. Figure 4A displays an overview of their relative quantification within effeminate and male samples. Within blood, we did not notice any significant alteration in their expression (P0,05). On the other hand, in urine, RNU-48 presented itself with a significant overexpression in females (PWhen it comes to age, we divided our 50 samples in 3 categories 20-40, 41-60 and over 60 years old. As it is shown in figure 4B, the relative quantification we achieved demonstrated no significant change in their expression profile (P0,05).4 Conclusion and future perspectivesMore than just a source of DNA, body fluids sole presence can have the most probative value. Hanson and colleagues introduced miRNA profiling as a reliable tool to identify body fluids such as blood, menstrual blood, semen, vaginal secretion and urine due to their tissue-specific pattern and stability when conditioned by degradation processes.Here we focused our attention in four miRNAs miR-127, miR-22 1, miR-222 and RNU-48. Soon enough miRNAs purity struck our attention when we notice that low value of 260/280nm ratio was associated with a poor degree of detection. When we upgraded our protocol the consequence reflected in a considerable decrease of the samples threshold.It would be irrefutably helpful to understand what threshold could affect miRNA profiling once, as it was shown, miRNA purity do affect intimately their quantification. It could even convey wrong outcomes once even miRNAs with high concentration within body fluids can appear with low concentration or totally inexistent.Our second result emphasised the importance of a normalisation gene. At first, we choose to use RNU-48 as our endogenous control but its behaviour within blood and urine make us reconsider our decision. RNU-48 is usually used as a reference gene due to its stable behaviour within samples however, our essay showed otherwise. Within the 4 miRNAs testes, RNU-48 was the one with a more pronounced vari ability within samples, which is opposed of what would be expected of a normalisation gene. Unexpectedly, miR-222 presented itself with the last(a) standard deviation between blood and urine. Furthermore, we studied its expression levels and compared them within age and gender and concluded that no significant alteration was noticeable (PAs stated earlier, normalisation genes are indispensable to sustain qRT-PCR results however, till date, no normalisation gene is universally acknowledged. This problem is reflected in our case, where one of the most used normalisation gene proved to be unsuitable for urine and blood miRNA analysis. This subject is a very sensitive point in miRNA profiling. There is why it is imperative to focus our future line of work towards finding a reliable normalisation gene before anything else.Our main goal was to define whether or not miR-127, miR-221 or RNU-48 could have the potential to be considered as biomarkers for body fluids identification. In this case, we could establish that all four have different expressional patterns in urine and blood (fig.5) however, to be considered as biomarker it would expected a major difference within body fluids which do not happen with our miRNAs considered for this essay. There is why we conclude that none of this miRNAs have the potential to be considered as a biomarker for body fluid identification.Conflict of interestNone.