TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR.

摘要: The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is most commonly-used platform due to its inexpensive nature robust chemistry, quantifying genes with low abundance or RNA samples extracted from highly restricted limited sources can be challenging because detection sensitivity limited. Here, we develop a novel effective touchdown (TqPCR) protocol by incorporating 4-cycle stage prior amplification stage. Using same cDNA templates, find that TqPCR reduce average Cq values for Gapdh, Rps13, Hprt1 reference 4.45, 5.47, 4.94 cycles, respectively, when compared conventional qPCR; overall value reduction three together 4.95. We further improve efficiency thus increase sensitivity. When Wnt3A-induced target mesenchymal stem cells analyzed, that, while both detect up-regulation relatively abundant Axin2, only lowly-expressed targets Oct4 Gbx2. Finally, demonstrate MRQ2 MRQ3 primer pairs derived mouse Tbp used validate RNA/cDNA integrity samples. Taken together, our results strongly suggest may efficiency. Overall, should advantageous over quantification, especially transcripts interest are lowly expressed, and/or availability total