Quantitative PCR (qPCR, also called Real-time PCR) is a popular technology for precise analysis of gene expression. It can be classified into two categories according to different methods, dye-based and probe-based, in which, dye-based method is more popular, convenient and less costly. Dye-based qPCR monitors real-time fluorescence of the dye binding to the double-stranded DNA to measure DNA amplification indirectly during each cycle. At a point where the fluorescence signal is distinctly detected over the background, Ct value (Cq value) can be determined. The obtained Ct values can be used to evaluate relative target abundance or calculate absolute target quantities in reference to an appropriate standard curve.

HotStart™ Universal 2X Green qPCR Master Mix provides superior specificity, robust amplification efficiency, ideal reproducibility and stability in quantifying target DNA or cDNA. It’s a 2X premix, taking advantage of a hot-start Taq polymerase combined with the antibody. Ideal Taq polymerase and suitable buffer will guarantee preferable specificity and high amplification speed. Green I in the mix which is a DNA intercalator will emit fluorescence when bounds to the double-stranded DNA amplified in each cycle and monitoring the fluorescence by the instrument allows for the indirect quantification of amplification products at real time. This reagent has special Specific ROX Reference Dye which is suitable for all qPCR instruments. Instead of adjusting the concentration of the ROX on different instruments, you can simply add primers and templates to amplify when formulating the reaction system.

However, dye-based qPCR has some limitations. Green I can intercalate into any double-stranded DNA, such as primer dimer or other undesired products, causing nonspecific products to emit fluorescence. To confirm the specificity of the product, following amplification, performing a melt curve analysis is necessary. A sharp peak around the annealing temperature in the melt curve analysis is ideal.