The detection of messenger RNA (mRNA) using reverse transcriptase PCR (RT-PCR) is becoming common practice for forensic body fluid identification. However, the degraded and scarce nature of RNA from forensic samples mean that mRNA transcripts are not consistently detected or remain undetected in practice. Conventional primer design for RT-PCR (and quantitative RT-PCR) includes targeting primers to span exon-exon boundaries or by having the primers on two separate exons, and satisfying common primer thermodynamic criteria.
Researchers at the Institute of Environmental Science and Research Limited, New Zealand have found that the conventional placement of primers is not always optimal for obtaining reproducible results from degraded samples. Using massively parallel sequencing data from degraded body fluids, they designed primers to amplify transcript regions of high read coverage, hence, higher stability, and compared these with primers designed using conventional methodology. They found this new primer design resulted in vastly improved detection of mRNA transcripts that were not previously detected or were not consistently detected in the same samples using conventional primers. The primers targeted to transcript stable regions (StaRs) are able to consistently and specifically amplify a wide range of RNA biomarkers in various body fluids of varying degradation levels.
- RNA stable regions (StaRs) identified from RNAseq (MPS) of degraded body fluids.
- StaRs are less degraded and hence ideal targets for forensic biomarker detection.
- StaR primers significantly outperform conventional primers in degraded samples.
- StaR primers consistently detect a wide range of forensic body fluid RNA markers.
- StaR primers perform in different body fluids at varying degradation levels.
(A) Male 6 week old buccal sample RNA sequencing read coverage map along the annotated reference of the saliva marker HTN3 and viewed in the sequence viewing software Geneious v5.6.7. Green represents coverage regions with reads in both directions; and yellow represents coverage regions with reads in one direction only. The black features depict the position of RT-PCR forward and reverse primers designed using conventional primer design methodology (A) and white features depict the position for RT-PCR StaR primers (B). X denotes the level of sequencing read coverage along the reference; Y denotes the annotated reference gene. (C) Electropherogram of a singleplex PCR amplification of cDNA from 6 week old buccal samples using HTN3 conventional primers (black arrow) and HTN3 StaR primers (white arrow). (D) Relative fluorescence units detected from singleplex PCR amplifications of different input cDNA volumes using HTN3 conventional and StaR primers, from fresh buccal (BuF) and 6 week old buccal (Bu6w) samples. (E) Relative fluorescence units detected from singleplex PCR amplifications of cDNA from 5 technical replicates of fresh buccal (BuF) and 6 week old buccal (Bu6w) using HTN3 conventional and StaR primers.