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

Abstract

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

Notch Signaling Augments BMP9-Induced Bone Formation by Promoting the Osteogenesis-Angiogenesis Coupling Process in Mesenchymal Stem Cells (MSCs)

“…PCR primers were designed by using the Primer3 Plus program [60]. The quantitative PCR analysis was carried out using our recently optimized TqPCR protocol [61, 62]. Briefly, the SYBR Green qPCR reactions (Bio-Rad Laboratories) were set up according to manufacturer's instructions.…”

A Blockade of IGF Signaling Sensitizes Human Ovarian Cancer Cells to the Anthelmintic Niclosamide-Induced Anti-Proliferative and Anticancer Activities

“…The qPCR primers were designed by using Primer3 program [64] and used to amplify human IGF1R: 5'-ATG ACA TTC CTG GGC CAG TG-3' and 5'-TAG CTT GGC CCC TCC ATA CT-3'. TqPCR were carried out by using the SYBR Green-based qPCR analysis on a CFX-Connect unit (Bio-Rad Laboratories, Hercules, CA) [65]. The qPCR reactions were done in triplicate.…”

Validation of RPS13 as a reference gene for absolute quantification of SIV RNA in tissue of rhesus macaques

“…RPS13 mRNA has been described as a medium abundance reference gene whose expression is tightly regulated via multiple redundant mechanisms (Jacob et al, 2013; Malygin et al, 2007; Zhang et al, 2015). Based on this property, several studies have validated and utilized RPS13 mRNA as a reference gene in relative gene expression studies, especially in oncology research (de Jonge et al, 2007; Dupasquier et al, 2014; Jacob et al, 2013; Rentoft et al, 2010; Zhang et al, 2015). …”