Utilization of Modified Touchdown qPCR to Enhance Sensitivity and Specificity for Genes with Restricted Expression and Low Optimum Temperature Primers

Background: Quantitative polymerase chain reaction (qPCR) is one of the most used methods to analyze gene expression. However, if the annealing temperature in the qPCR cycle is lower than 55°C, nonspecific amplification may occur. On the other hand, some sets of primers can only function optimally at the annealing temperature below 55°C. Thus, a protocol allowing both specific amplification and large yield in low annealing temperature is needed. Methods: qPCR on the target ribosomal protein L13a (rpl13a) and natriuretic peptide A (nppa) from samples of zebrafish embryos and larvae was performed in triplicates using three different protocols: routine qPCR, gradient qPCR, and modified touchdown qPCR. The cycle of quantification (Cq) value and melt peak data were compared between the routine or gradient qPCR and the modified touchdown qPCR. Results: For rpl13a that was adequately and specifically amplified by both routine and touchdown qPCR, modified touchdown qPCR decreased the average Cq by 3.68-4.66 (P < 0.05). For nppa, a gene with restricted expression that could only be amplified substantially but nonspecifically at the annealing temperature of 50°C, modified touchdown qPCR increased the amplification specificity by forming only one melt peak during melt curve analysis and yielding a substantial amount of amplicon with the Cq of 22.48-22.51. Conclusions: The modified touchdown qPCR with low final annealing temperature was able to produce specific amplification with better yield and detection sensitivity. This method is especially useful for primer sets with low optimum annealing temperature.