Optimizing the efficiency of a probe-based duplex qPCR assay for accurate detection of normalized PI4KA gene copy number

Abstract

Quantitative PCR (qPCR) is a powerful molecular tool, and its capability to multiplex enhances its utility. However, multiplexing can reduce PCR efficiencies compared to monoplex reactions. This study aims to establish comparable PCR efficiencies for a probe-based duplex qPCR assay that quantifies PI4KA gene copy number relative to the reference gene SHANK3. Three standard curves were generated for two monoplex reactions and duplex assay with a 10-fold DNA dilution series (100 ng, 10 ng, 1 ng, and 0.1 ng) to evaluate the efficiencies in the PCR. All reactions were duplicated on the Bio-Rad CFX96 Touch™ system with previously optimized conditions. Reaction efficiencies were calculated as 10-1/slope−1 using the Bio-Rad CFX Maestro application. Each monoplex reaction demonstrated high efficiency (E=80%-110%) and a reliable correlation coefficient (R²≥0.98). However, in the duplex response, the efficiency of SHANK3 amplification was observed to be compromised (EPI4KA=108.5%, R2PI4KA=0.98, ESHANK3=149.5%, R2SHANK3=0.86). Thus, to enhance SHANK3 amplification in the duplex, primer concentrations for PI4KA (0.2-0.4 μM) were reduced relative to SHANK3 (0.4-0.8 μM) in various combinations. Based on the results, the primer concentrations that produced the lowest combination of Cq values, PI4KA Forward/Reverse 0.2 /0.2 μM (Cq=22.4+0.4) and SHANK3 Forward/Reverse 0.4 μM/0.8 μM (Cq=24.7+0.2) were selected for reconstructing the duplex PCR. The regenerated standard curve for the optimized duplex PCR showed amplification efficiencies of 98.6% (R2=0.99) for PI4KA and 99.4% (R2=0.90) for SHANK3. After the final optimization of the duplex PCR, the gene dose values obtained for the positive (i.e. PI4KA gene deleted; ≃0.5) and control (healthy; ≃1) samples confirmed their accuracy in detecting normalized gene doses. Although optimizing the PCR efficiency of the duplex qPCR provided a robust assay for detecting PI4KA copy number, further validation of the assay for precision and sensitivity is recommended before its clinical use.