qPCR Primer Pairs

45 pairs of gene-specific qPCR primers were experimentally validated by SYBR Green method using first-strand cDNA and ddH2O as template. There were no non-specific amplification and primer-dimers produced by melting curve analysis and agarose gel electrophoresis detection. We used a dilution series (10-fold dilutions) of a purified plasmid as templates to construct standard curves for primer by SYBR Green-based real-time RT-PCR. We drew the standard curve by SYBR Green method in the qPCR experiment. The result showed that the qPCR Primers detectable limit is less than 100 copies and the amplification efficiency is between 90% ~ 105%. a. Amplification curves of 45 primer pairs in cDNA. (cDNA and ddH2O as templates);b. Melting curves of 45 primer pairs corresponding with a; c. Agarose gel electrophoresis validation result corresponding with a, the odd number lane is cDNA template detection, the even number lane is the corresponding NTC (No Template Control) results; d. Statistical graph of amplification efficiency about 45 primer pairs with a dilution series plasmid as templates; e. Amplification curves of SDHA standard curve; f. Standard curve of SDHA, the amplification efficiency is 99.0%.