Development of a Standard Curve for Quantification of LINE-1 Methylation on Real-time PCR Systems

Abstract

: Real-time PCR is currently one of the most powerful molecular approaches and is widely used in molecular biology diagnostics. Today, many real-time PCR systems differ in the thermal block, the excitation source, the fluorescence detection system, and the data analysis software. Therefore, using different systems may affect the detection limit of biological samples. Our study aims to optimize the threshold to ensure the amplification efficiency (E) and correlation coefficient (R2) of the standard curve used to quantify LINE-1 methylation on three real-time PCR systems, including: Applied Biosystems™ 7500, QuantStudio™5 Dx; and abCyclerQ (AIT Biotech). We determined a proper threshold to build a standard curve on the three systems, which ensures the correlation coefficient (R2) and the amplification efficiency (R2 > 0.999, E ≥ 95%). Therefore, it is possible to use interchangeably in Applied Biosystems™ 7500, QuantStudio™5 Dx, and abCyclerQ (AIT Biotech) in LINE-1 methylation analysis.