Identification of Body Fluid Stains Using Real-time RT-PCR: Discrimination Between Salivary, Nasal, and Vaginal Secretions

Abstract

In a criminal investigation, it is important to clarify the origin of the extracted DNA from available biological samples. In recent years, a new approach for the identiˆcation of body ‰uid stains has been demonstrated and involves a comparison of speciˆc mRNA expression levels. Here, we used real-time RT-PCR to examine the expression levels of STATH, HTN3, MUC4, and ESR1 genes from salivary, nasal, and vaginal secretions which are often thought to be di‹cult to discriminate between. STATH was expressed at high levels in all salivary (dCt＝1.27±3.18, n＝ 10) and all nasal secretions (dCt＝-0.99±3.94, n＝8), and at low levels in 3 of the 10 vaginal secretions (dCt＝16.34-17.47). HTN3 was expressed at high levels in all salivary secretions (dCt＝0.67±3.08). MUC4 was expressed at relatively high levels in all vaginal secretions (dCt＝4.97±2.36, n＝10), and at moderate levels in 5 of the 10 salivary secretions (dCt＝8.55-10.40) and in 4 of the 8 nasal secretions (dCt ＝6.56-8.23). ESR1 was expressed at relatively high levels in all vaginal secretions (dCt＝5.79±2.90), but no expression was found in salivary and nasal samples. These results indicate the possibility for speciˆc discrimination between various body ‰uids. To test the practicality of this, gene sensitivity and stability were examined in saliva, semen, and blood stains. Several genes, including ACTB, STATH, HTN3, PRM2, SEMG1, and HBB, were shown to be detectable even in small volume (0.1 mL) stains and were also expressed in 1-year-old stains. mRNA stability was dependent on favorable environmental factors, such as dryness and shading. In addition, the successful detection of target genes from mixed stains and simulated casework samples might promise the utility of this assay. The mRNA assay therefore appears to be a novel tool for body ‰uid identiˆcation from biological samples.

Current Methods for Body Fluid Identification Related to Sexual Crime: Focusing on Saliva, Semen, and Vaginal Fluid

“…In particular, real-time RT-PCR using the mRNA expression levels of salivary-specific proteins has been reported [37][38][39]. We have also confirmed the usefulness of the STATH and HTN3 genes [40,41] and performed various studies to introduce the method into practice [42][43][44]. In addition, methods focusing on microRNA [45][46][47][48][49][50] and DNA methylation [51][52][53][54][55][56] have been actively explored for saliva identification.…”

“…As for saliva and semen, many studies have been conducted using real-time RT-PCR [38,90,91]. We have also performed similar studies [35,41] and have revealed the usefulness of multiplex detection specifically targeting ESR1, SERPINB13, KLK13, CYP2B7P1, and MUC4 genes [92,93] for vaginal fluid identification. Methods using microRNA [45,46,48,49] and DNA methylation [52][53][54][55] are also being examined.…”

“…Furthermore, methods using Raman spectroscopy [111] or Fourier-transform infrared spectroscopy [112] have been reported as non-destructive methods for analysis that do not even consume a minute amount of residual sample, although further studies are required before they can be practically applied. [37][38][39][40]45,46,[48][49][50][51][52][53][54][55][56]76,77] Vaginal fluid Immunodiffusion Immunoelectrophoresis [34] Papanicolaou staining Lugol's staining [79][80][81][82] mRNA microRNA DNA methylation [35,38,41,45,46,48,49,[52][53][54][55][90][91][92][93] Vaginal bacteria [83][84][85][86][87][88][89] GC-MS LC-MS [94,…”

Application of mRNA Expression Analysis to Human Blood Identification in Degenerated Samples that were False‐negative by Immunochromatography,,

“…The analysis of body fluid-specific mRNA expression levels has recently been reported as a promising tool for the identification of body fluids in biological stains (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In 2005, Juusola and Ballantyne reported multiplex mRNA profiling for the identification of body fluids using agarose gel electrophoresis and capillary electrophoresis format (8).…”

Detection of proline-rich proteins for the identification of saliva by enzyme-linked immunosorbent assay

“…Therefore, by cut-off absorbance value at 0.2 the PRH1/2 ELISA is useful for saliva identification, despite aging and mixing of samples, which could cause digestion and denaturation of proteins, reinforcing its potential value as a method for forensic saliva identification. HTN3 mRNA has previously been shown to be a good marker for saliva identification [5,22]. However, the protein-based method for detecting HTN3 has not been reported because the useful antibody for forensic investigation cannot be available at present.…”