Development and validation of a non-invasive RT-qPCR protocol for Nrf2 mRNA quantification in human tear fluid: A...

Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master transcriptional regulator of cellular antioxidant responses and has been implicated in ocular surface diseases including keratoconus and dry eye. However, validated methods for non-invasive assessment of Nrf2 pathway activity in human tears are lacking. Here we describe the development and validation of an RT-qPCR protocol for quantifying Nrf2 mRNA in tear fluid collected via Schirmer strips. The method employs modified TRIzol extraction optimized for ultra-low volumes (10-15 μL), SYBR Green chemistry with exon-spanning primers, and RPL13a as a validated reference gene for tear fluid matrix. Validation in 35 healthy adults according to MIQE guidelines demonstrated: primer efficiencies of 98.2% (Nrf2) and 101.5% (RPL13a) with R² > 0.99; linear dynamic range spanning 4 log₁₀ units; limit of detection at Ct ∼35; single-peak melting curves confirming specificity; and intra-assay coefficient of variation <15%. The detection rate was 74.3% (26/35) with mean Ct of 25.2 ± 1.8. This is the first validated protocol for Nrf2 mRNA quantification in human tear fluid, bridging a critical methodological gap between bench-based redox research and clinical biomarker application. The protocol is readily adaptable for other transcriptional targets and has potential applications in disease stratification and pharmacodynamic monitoring of Nrf2-activating therapeutics.