Age determination has long been one of the most important goals of forensic scientists and the biological age of an individual is an advantageous biometric that may be open to molecular genetic analysis. Messenger RNA (mRNA) may provide the necessary specificity, sensitivity and automation capabilities that modern forensic biology laboratories require for cellular origin identification . The current study aimed at determining the age of individuals based on differences in gene expression.
The study focused on three skin-specific mRNA markers, namely, Corneodesmosin (CDSN), Loricrin (LOR) and Keratin 9 (KRT9). The three target genes are involved in the differentiation or maintenance of the keratinization and cornification of the skin. KRT9 belongs to the superfamily of intermediate filament proteins that are expressed in all different epithelial cell types. About 20 human epithelial keratins exist, and each function as an important building block of the cytoskeleton of epithelial cells; also, each epithelial keratin has its own specific function or timing in the cellular dynamics. They are expressed, mostly in pairs or subsets, during terminal skin cell differentiation, in the different stages of development and in different epithelia . A type I keratin, KRT9, is expressed only in the suprabasal cells of the epidermis and has previously been found to be specifically expressed in palmar and plantar skin 
CDSN, a 52- to 56-kDa basic glycoprotein, is specific to the cornified epithelia and the inner root sheath of hair follicles . LOR is initially expressed in the granular layer of the epidermis during cornification and comprises about 80% of the total protein mass of the cornified envelope . Both CDSN and LOR are involved in the assembly of the epidermal cornified cell envelope, with CDSN mainly detected in uppermost spinous and granular layers . Beta-actin (ACTB), the reference gene used in the study was used for normalizing expression signals of skin-targeted mRNA markers. It was chosen from five commonly used genes (β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin B (PPIB) and Ubiquitin C (UBC)) , which were all tested for ubiquitous expression across forensically relevant samples. Of all reference candidate genes, ACTB has been reported to have the least variation in salivary epithelial cell . Actins are highly conserved proteins that are involved in cell motility, structure and integrity. ACTB is a major component of the contractile apparatus and is also one of the two non-muscle cytoskeletal actins.
The RNA concentration and yield were very low across all samples. It was suggested that this was due to ribonucleases present in saliva, which could hinder analysis of RNA in the saliva . Recent studies suggested that the method which uses a saliva-specific RNA extraction kit always produces better RNA concentration and yield characteristics as it contained a protecting solution which stabilizes salivary RNA . It therefore was suggested that the low RNA concentration of the saliva samples might be due to the use of a general RNA extraction kit and not a saliva-specific RNA extraction kit. It was observed that there was a significant (p < 0.01) correlation between RNA quality and RNA yield, and this can be interpreted as the less contaminated the sample is, the greater the yield expected.
The CDSN gene was detected in all the sampled age groups. Though the age group 16–20 had the highest expression of CDSN among the three age groups, there was no significant difference (p > 0.05) in the expression of the gene among the three age groups. The expression of the CDSN gene in the control was significantly different (F = 17.08, p ≤ 0.05) from its expression in all three age groups. The LOR gene was lowly expressed across all age groups used in the study. The expression of the gene did not significantly differ (p > 0.05) between the control and age group 26–30, but they were however significantly higher (F = 36.47, p ≤ 0.05) than the expression of the gene in both 16–20 and 21–25 age groups. The KRT9 gene was expressed only in age groups 16–20 and 26–30 and the expression of the gene did not significantly differ (p > 0.05) between these age groups. The gene was not detected in the control and 21–25 age groups.
While both CDSN and LOR genes were detected in all age groups which agrees with a study carried out by Visser et al. , KRT9 was only detected in 16–20 and 26–30 age groups. Among the three genes, CDSN had the highest expression across the three genes. CDSN and LOR have high sensitivity for human skin epithelial cells , and this can be confirmed by the presence of CDSN and LOR, though low, in all saliva samples across the three age groups, due to the fact that buccal cells which are present in the saliva have strong similarity with epithelial cells. Gomes et al.  reported that preliminary results suggest a probable lower sensitivity of detection for KRT9 in the analysed skin tissues, and this could account for the absence of KRT9 in the second age group (21–25).
This study was designed to define the phenotypic differences between young adults within different age groups in relation to mRNA expression levels of certain genes. Individual variation in gene expression may likely be a result of the collection of all relevant genetic influences . Although RNA concentration was low and the expression values of the genes were low and could not be used in comparing the expression levels among the three age groups, it can be concluded that the three mRNA markers CDSN, LOR and KRT9, as well as the ACTB reference mRNA marker analysed via the described qPCR assays, are suitable for identifying epithelial cells in saliva.