Evaluation of 18 S rRNA as an endogenous control in OpenArray® plasma RNA qPCR workflows

Abstract

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.