Evaluation of Stem-Loop Reverse Transcription and Poly-A Tail Extension in MicroRNA Analysis of Body Fluids

Title:Evaluation of Stem-Loop Reverse Transcription and Poly-A Tail Extension in MicroRNA Analysis of Body Fluids

Volume: 3 Issue: 3

Author(s): Hannah Dunnett, Dieudonne van der Meer and Graham A. Williams

Affiliation:

Keywords: Blood, forensic genetics, MicroRNA, poly-A tail extension, qPCR, saliva, stem-loop reverse transcription.

Abstract: 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.

Cite this article as:

Dunnett Hannah, van der Meer Dieudonne and A. Williams Graham, Evaluation of Stem-Loop Reverse Transcription and Poly-A Tail Extension in MicroRNA Analysis of Body Fluids, MicroRNA 2014; 3 (3) . https://dx.doi.org/10.2174/2211536604666150121000603