Evaluation of stem-loop reverse transcription and poly-A tail extension in microRNA analysis of body fluids

MicroRNA has been demonstrated to be a viable tool for body fluid identification purposes in forensic casework. Stem-loop reverse transcription (slRT) is regularly used for cDNA synthesis from mature miRNA, along with poly-A tail extension. Both have been used in a forensic context, but no direct comparison has been carried out. It has also not been shown whether poly-A tail extension can be used upon DNA extracts, as previously shown with slRT. Blood and saliva samples were collected and underwent DNA extraction with or without on-column DNA digestion. All samples were then aliquoted and underwent slRT and poly-A tail extension separately. qPCR was then conducted targeting microRNA markers hsa-miR-451 and hsa-miR-205. It was shown that the DNA digestion step did not affect the ability to differentiate between blood and saliva. It was also shown that this differentiation was possible using poly-A tail extension, and that poly-A tail extension exhibited more amplification than slRT. So whilst the choice of slRT and poly-A tail extension for the purpose of forensic body fluid identification is not critical, it may be best to use poly-A tail extension, particularly where there are low traces of sample.