Journal Article Epublish
PubMed: 42801352 DOI: 10.1007/s11033-026-12820-9
Evaluation of 18 S rRNA as an endogenous control in OpenArray ® plasma RNA qPCR workflows.
Mol Biol Rep 53(1)
Authors: Habib Francis, Andrzej S Januszewski, Abubakar Mangani, Fahmida K Ema, Anandwardhan A Hardikar, Mugdha V Joglekar, Michael L H Huang, David R Sullivan, Ronald C W Ma, Sanjeev Galande, Val Gebski, R John Simes, Alicia J Jenkins, Mark P Molloy, Anthony C Keech
Affiliations
- Sydney Clinical Trials Centre, The University of Sydney, Sydney, Australia.
- Sydney Clinical Trials Centre, The University of Sydney, Sydney, Australia.
- Sydney Pharmacy School, The University of Sydney, Sydney, Australia.
- Western Sydney University School of Medicine, Campbelltown, Australia.
- Baker Heart and Diabetes Institute, Melbourne, Australia.
- Royal Prince Alfred Hospital, Camperdown, Australia.
- Department of Medicine and Therapeutics and Li Ka Shing Institute of Health Sciences, Faculty of Medicine, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China.
- Shiv Nadar Institution of Eminence, Tehsil Dadri, Gautam Buddha Nagar, Uttar Pradesh, India.
- Sydney Clinical Trials Centre, The University of Sydney, Sydney, Australia. Baker Heart and Diabetes Institute, Melbourne, Australia.
- Bowel Cancer and Biomarker Research Laboratory, School of Medical Sciences, Kolling Institute, The University of Sydney, St Leonards, Australia.
Abstract
BACKGROUND
Reliable qPCR analysis depends on stable endogenous controls, particularly in high-throughput pre-amplification workflows where technical bias may arise. The suitability of highly abundant 18 S rRNA as a reference gene in multiplex OpenArray ® platforms remains debated. This study evaluated the stability of 18 S rRNA, its impact on co-amplified targets, and comparability between archived and newly manufactured primer pools.
METHODS
Plasma RNA from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial was analysed using TaqMan OpenArray ® following reverse transcription and pre-amplification. Paired samples (n = 23) were pre-amplified with primer pools with and without 18 S rRNA and compared across four target genes. Stability of 18 S rRNA was assessed in 789 samples across 17 runs. Agreement between archived and new primer pools was evaluated in 774 paired samples using Wilcoxon signed-rank and Bland-Altman analyses.
RESULTS
18 S rRNA showed stable detection (median Ct 14.5, IQR 13.4-16.3) with low inter-run variability (CV 3.9%). Inclusion of 18 S rRNA during pre-amplification did not significantly alter Ct values of co-amplified targets (median differences 0.07-0.52 cycles; p > 0.05), with minimal overall bias (-0.18 Ct). Archived and newly manufactured primer pools showed a small but significant Ct shift, with lower values in the new pool (bias 0.53 Ct; p < 0.0001), although variability between paired measurements was wide.
CONCLUSION
18 S rRNA is a stable technical control in plasma OpenArray ® workflows and did not measurably bias co-amplified targets during pre-amplification. Archived and new primer pools showed only minor systematic differences and remained broadly comparable at the group level.
MeSH
Genes, Bias, Genes, Rrna, Workflow, Plasma