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Optimal small-molecular reference RNA for RT-qPCR-based body fluid identification

Shuntaro Fujimoto1, Sho Manabe1, Chie Morimoto1

  • 1Department of Forensic Medicine, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.

Forensic Science International. Genetics
|September 2, 2018
PubMed

Insights

This study identifies optimal reference RNAs for microRNA-based body fluid identification in forensics. 5S-rRNA is the best single reference, with combinations enhancing normalization for accurate forensic analysis.

Area of Science:

  • Forensic Science
  • Molecular Biology
  • Genetics

Background:

  • MicroRNA (miRNA)-based body fluid identification (BFID) is crucial in forensic science.
  • Accurate normalization using stable reference RNA is essential for reliable BFID via reverse transcription-quantitative PCR.

Purpose of the Study:

  • To evaluate candidate reference RNAs for robust normalization in forensic BFID.
  • To identify the optimal reference RNA or combination of RNAs for body fluid identification.

Main Methods:

  • Assessed sample quality using RNA integrity number.
  • Evaluated reference RNA expression stability in four body fluids using NormFinder and BestKeeper.
  • Selected optimal reference RNAs using the RankAggreg package in R.

Main Results:

  • RNA integrity was low despite pristine samples.
  • 5S-rRNA was identified as the optimal reference RNA for BFID.
  • Combinations of 5S-rRNA with miR-92a-3p and/or miR-484 improved normalization quality.

Conclusions:

  • Established 5S-rRNA as a superior reference RNA for forensic BFID.
  • Demonstrated that combining 5S-rRNA with specific miRNAs enhances normalization accuracy.
  • Findings support the development of more stringent small RNA-based BFID systems.
Keywords:
Body fluid identificationNormalization in quantitative PCRSmall-molecular reference RNAmicroRNA

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