MESSENGER RNA BASED SKIN IDENTIFICATION USING SKIN SPECIFIC MARKERS FROM FINGERPRINT IMPRESSIONS

The study of forensic science may be complex especially in the areas of nucleic acids and trace evidence. Oftentimes, forensic scientists recover minute quantities of biological material from scenes therefore the need to generate the genetic profiles and identify the source of the material. The aim of this study is to determine the expression levels of skin-specific gene markers – Loricrin (LOR), Corneodesmosin (CDSN) and Keratine 9 (KRT9), on fingerprint impressions of individuals of specific age group and genders. Thumbprints were collected using labelled frosted microscope glass slides and cello tapes. Messenger RNA was extracted from the samples, converted to cDNA and amplified by qPCR with its specific primer sequences. In males, RNA yield was higher in the slide (26.96±8.68) compared to cello tape (18.32±4.52) while the reverse was the case in the females. Across gender, RNA purity and yield were higher in males than females. In males, KRT9, LOR and CDSN genes were more expressed using frosted slide (37.03±0.77, 40.46±2.66, 35.62±2.82) compared to cello tapes (35.33±0.3, 32.11±0.5, 35.28±0.86) respectively. In females, LOR, CDSN and ACTB genes were more expressed using frosted slides (36.26±0.8, 37.37±0.58, 26.63±0.12) compared to cello tapes (35.52±1.01, 35.57±3.22, 26.57±1.18) respectively. Across gender, LOR, KRT9 and ACTB genes were more expressed in males than females while CDSN was more expressed in females than males. The expression levels of CDSN and ACTB genes were significantly (p≤0.01) correlated. This study shows that mRNA markers LOR, CDSN and KRT9, analysed via the RT-qPCR assays, are highly suitable for identifying skin cells even in small traces.


INTRODUCTION
There is a strong trend in molecular forensics for the development of alternative techniques to identifying the cellular origin of biological samples and trace evidence collected at crime scenes (HALL et al., 2013). Information about the possible origin of biological samples of forensic traces is mostly ascertained via protein-based presumptive testing and DNA analysis. Messenger RNA-profiling has however emerged as an alternative strategy to

Ribonucleic acid (RNA) extraction
Ribonucleic acid (RNA) was extracted from the samples collected using a Pinpoint slide RNA isolation system 1 kits (Zymo research®) following the manufacturer's instructions. The Pinpoint slide RNA isolation system 1 kit procedure involved two steps which were Pinpoint fractionation to recover fingerprint samples from glass slides and cut cello tapes and RNA extraction for total RNA recovery.

Complementary DNA (cDNA) synthesis
Complementary DNA (cDNA) synthesis was carried out using the LunaScript RT SuperMix Kit (NEB #E3010). The cDNA synthesis reaction was prepared by adding 4 µl of LunaScript RT SuperMix (1X) to 10 µl of the RNA sample and made up to 20 µl with 6 µl of nuclease-free water. The reactions will then be incubated with primer annealing for 2 minutes at 25 °C, cDNA synthesis for 10 minutes at 55 °C and heat inactivation for 1 minute at 95 °C.

Statistical analysis
Data generated were analysed using IBM SPSS Version. 26 (IBM SPSS Inc., USA). Analysis of variance (ANOVA) was used to compare group means and mean differences were separated using Duncan Multiple Range Test at 5 % level of significance. Graphs were plotted using GraphPad 8.0.1 software. The comparative Ct method (2 -∆∆Ct method) was used to analyse the expression level of the target genes according to LIVAK and SCHMITTGEN (2001) and RAO et al., (2013).

Primers used for the study
The primer sequences for the target genes and reference gene were designed on Primer blast and validated on Netprimer and Oligo analyzer. The primer sequences are shown in Table  1.

Ethical clearance
Informed written consent was obtained from all the study participants, and the study protocol was approved by the Health Research ethics committee of the College of Medicine, University of Lagos (CMULHREC Number: CMUL/HREC/O668/19).

RESULTS
In Table 2, KRT9, LOR and CDSN genes were more expressed in samples taken on a frosted slide (37.03 ± 0.77, 40.46 ± 2.66, 35.62 ± 2.82) than cut cello tapes (35.33 ± 0.3, 32.11 ± 0.5, 35.28 ± 0.86) amongst the male participants respectively. However, the reverse was the case for the ACTB gene which showed that the samples taken on cut cello tapes expressed more of this gene (27.05 ± 0.11) than the samples collected on frosted slides (26.94 ± 0.67). Amongst the female participants, LOR, CDSN and ACTB genes were more expressed in samples taken on frosted slides (36.26 ± 0.8, 37.37 ± 0.58, 26.63 ± 0.12) than those taken on cut cello tapes (35.52 ± 1.01, 35.57 ± 3.22, 26.57 ± 1.18) respectively. However, the reverse was the case for the KRT9 gene which showed that the samples taken on cut cello tapes expressed more of this gene (35.4 ± 2.2) than the samples collected on frosted slides (34.76 ± 1.24). Across gender, LOR, KRT9 and ACTB genes were more expressed in males than females while CDSN was more expressed in females than males. At 260/280 absorbance ratio (Table 3), samples which were taken on cut cello tapes yielded purer (1.96 ± 0.24) RNA than those collected on frosted slides (2.07 ± 0.21) in male while slide samples yielded purer (1.73 ± 0.02) RNA than cello tape samples (1.68 ± 0.13) in females. Also, RNA yield was higher in male slide samples (26.96 ± 8.68) than cello tape samples (18.32 ± 4.52) and the reverse was the case in the females. Across gender, RNA purity and yield were higher in males than females.  Table 4 shows that there was no significant difference in Cq values of KRT 9, CDSN and the ACTB gene across gender and type of surfaces used. There was a significant difference (p ≤ 0.05) between the expression of the LOR gene in samples collected on slides and cello tapes among the males. Also, there was a significant difference (p ≤ 0.05) between the expression of the LOR gene in female samples collected cello tapes and male samples taken on slides. Table 5 shows that there was no significant difference in spectrophotometric values of all extracted RNA samples at 260/280 and 260/230 absorbance ratio and in the concentration values across gender and type of surfaces used.
In Table 6, Pearson correlation analysis showed that there was a correlation between the expression levels of CDSN and ACTB genes at 0.01 level of significance. However, the ACTB expression level did not correlate with the expression levels of KRT9 and LOR genes (p˃0.05). Also, RNA Purity (260/230) correlated with concentration at 0.01 level of significance.
In Table 7, Spearman's rho correlation analysis showed that there was a correlation between the expression levels of CDSN and ACTB genes at 0.05 level of significance. Also, RNA Purity (260/230) correlated with concentration at 0.01 level of significance.
In Table 8, Kendall's tau-b correlation analysis showed that there was a correlation between the expression levels of CDSN and ACTB genes at 0.05 level of significance. Also, RNA Purity (260/230) correlated with concentration at 0.01 level of significance.      The inability to link the DNA profiles to the tissue source and prove that the DNA originated specifically from skin cells can lead to challenges against DNA evidence in court. Proper identification of the biological materials present forensic samples is important to the investigation, personalization and prosecution of a criminal offense and a misrepresentation of the nature of the evidence can have undue influence on the perception of the circumstance of the crime. Currently, there are no routinely validated methodologies used for the identification of skin epithelial cells. Utilization of mRNA profiling for the identification of forensically relevant biological fluids such as blood, semen, saliva, vaginal secretions and menstrual blood has been documented BALLANTYNE, 2003, 2005;HAAS et al., 2009). Hence, the need for the development and validation of an mRNA assay that targets highly overexpressed genes in skin in relation to other cell types usually encountered in crime scenes is crucial.

DISCUSSION
From the study, RNA yield ranged from 7.2 -55.1 ng/µl in males and 6.8 -33.1 ng/µl in females. Also, the RNA yield was higher in males than females. This agrees with the report of HANSON et al., (2011), who reported that the total input RNA should range from (5 -25 pg) to be exquisitely sensitive to detect skin traces in evidentiary items.
KRT9, ACTB and LOR markers were more expressed in males than females while CDSN was more expressed in females than males. Across gender, KRT9, CDSN and LOR markers were more expressed than ACTB. These three target genes KRT9, CDSN and LOR were moderately expressed in the fingerprint impressions taken on frosted slides and cut cello tapes. Hence, this makes them suitable markers for skin cell identification and this agrees with the report of VISSER et al., (2011), documented that CDSN, LOR and KRT9 genes showed strong over-expression in skin samples relative to samples from forensic body fluids and thus make them suitable markers for skin identification. However, HALL et al., (2013), reported that they failed to detect KRT9 in any of the donors' soles (or arms) and only one participant indicated the presence of this marker in their palm. Hence, they suggested that these markers must be used with caution in the identification of skin cells in forensic samples. Additionally, GOMES et al., (2011) documented that they encountered detection problems for the KRT9 gene which they linked to non-stringency of the pair of primers used and because preliminary results suggest a probable lower sensitivity of detection for KRT9 in the analysed skin tissues. LOR had the highest expression level (40.46 ± 2.66) and this agrees with the findings of HALL et al., (2013), who reported that LOR was also detected most frequently in the face and palm, as well as the leg; 61 %, 57 % and 64 % respectively. GOMES et al., (2011), also reported that LOR was a more stable and sensitive mRNA marker for human skin identification thus, CDSN and KRT9 should not be used alone, but concomitant with LOR.
The difference between the expression levels of the LOR gene in male and cut cello tape samples was significant (p ≤ 0.05). Also, a significant difference was observed between the female tape and male slide samples (p ≤ 0.05). CDSN expression level correlated with the expression of ACTB (p ≤ 0.01) but did not correlate with LOR and KRT9. HALL et al., (2013), documented that CDSN was observed to be the most sensitive marker, with the greatest expression rates detected in swabs taken from the forehead.

CONCLUSION
Messenger RNA (mRNA) profiling has been reported to be a good method used to identify most forensic materials obtained from forensic scenes. The mRNA markers LOR, CDSN and KRT9, analysed via the described qPCR assays, are highly suitable for identifying skin cells, including small traces of skin materials. Although the approach for skin identification introduced here can be improved upon to be more informative when applied on its own, however, it is recommended that the inclusion of other skin-targeted mRNA markers in multiplex systems, targeting other forensically relevant cell types should be used for further studies.