The applications of RNA analysis on identification of body fluids and dating plucked hairs

The aim of this study is to explore several possible uses of RNAs in forensic science, including body fluid identification using LAMP (loop-mediated isothermal amplification) technology and dating the plucked hair by real-time PCR. A novel application of real-time reverse transcription LAMP to identify the presence of a specific body fluid using blood and saliva is reported as a proof-of-concept model in this study. In comparing with other recently developed methods of body fluid identification, the RT-LAMP assay is more rapid and requires only one simple heating-block maintained at a single temperature, circumventing the need of dedicated equipment. The 18S rRNA locus was used as the internal control, and haemoglobin beta (HBB) and histatin 3 (HTN3) as the blood-specific and saliva-specific marker respectively. The data showed that the specificity and the limit of detection was a minimum of 10-5 ng total RNA for detection of both 18S rRNA and HBB, whereas it was 1.25ng total RNA for detection of HTN3. The detection of RT-LAMP products was performed by separation of the products using gel electrophoresis and collecting the fluorescence of calcein. The data collected indicated complete concordance with the body fluid tested regardless of the detection method used. This is the first application of real-time RT-LAMP to detect body fluid-specific RNA and demonstrates the use of this method in forensic biology.The determination of the time since a hair was plucked was estimated by the analysis of 18S rRNA and ß-actin mRNA using real-time PCR. The difference of average Cq value (dCq between the Cq values of 18S rRNA and ß-actin mRNA was monitored over a period of 8 weeks. The data presented that the age of the plucked hair could be approximately estimated using a liner polynomial: the time since the hair was plucked (TSP)= 0.168 × dCq + 13.406 (r=0.99) with a limitation of 21 days. It was also noticed that some factors, such as the uncertainty of the actual phase of the plucked hairs, chemicals used for hair dye or perm, and other uncontrolled environmental conditions, may impact the estimation