Optimizing qPCR Annealing Temperatures for Cancer-Related lncRNAs

Objective: To determine the optimal annealing temperatures to detect copy number variation and expression levels of specific cancer-associated long non-coding RNAs (lncRNAs), to improve the accuracy of clinical tests in precision medicine. Methods: Gradient qPCR analysis was performed to identify the optimal annealing temperatures for the detection of lncRNAs. These lncRNAs were H19, CCAT1, HOTAIR, NEAT1, MALAT1, PVT1, GAS5, BANCR, UCA1, HULC, and MEG3. Results: Optimal qPCR annealing temperatures for the detection of lncRNA expression and copy number variations were determined as follows: 62.4°C for PVT1 and BANCR; 60°C for H19, CCAT1-b, HOTAIR, NEAT1, MALAT1; 58°C for GAS5 and HULC; 56.7°C for CCAT1-a, UCA1; and 56°C for MEG3. Conclusion: In this study, we determined optimal annealing temperatures for some lncRNAs, which is crucial for the precision and accuracy of qPCR used to identify lncRNA expression and copy number changes. These results confirm the optimization of lncRNA analysis by determining optimal annealing temperatures, which is essential for the precision and accuracy of qPCR.