Performing body fluid identification with microRNAs using capillary electrophoresis

Abstract

MicroRNAs have a potential to be ideal forensic markers due to their small size (∼22nt), high abundance per cell, and sensitive and specific detection. Thousands of microRNAs are present in biological material and they are suitable for body fluid identification (BFID). Their advantageous properties increase the chances of successful analysis from challenged crime scene samples. In addition, it has been demonstrated that informative microRNA expression levels can be obtained from common DNA extracts. Following an earlier pilot project on a single stream process with the integration of microRNA analysis into a DNA profiling multiplex, progress on this line of research is now presented. A panel of 8 microRNAs (hsa-miR-10a, -16a, -135a, -142, -203a, -205, -451a and -1260b) has been identified to allow differentiation between blood, saliva, semen and vaginal material. Here the analysis of the BFID markers using capillary electrophoresis (CE) on ABI’s 3130 genetic analyser is presented. The markers are reverse transcribed using a multiplex stem-loop reverse transcription, followed by PCR with ROX-labelled universal reverse primer for the detection of cDNA.It is shown that – after careful optimization – BFID microRNA analysis using CE has similar discriminatory power as using qPCR in singleplex reactions and is therefore a viable technique for BFID. Multiplexing these markers is a next step that can result in a single test for BFID with the advantageous properties of microRNAs.