ArticlePloS one2015
TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 82 papers.
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Who cites it
82 citing papers in PubMed, 118 citations in OpenAlex.
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- Effective Bone Tissue Fabrication Using 3D-Printed Citrate-Based Nanocomposite Scaffolds Laden with BMP9-Stimulated Human Urine Stem Cells.ACS applied materials & interfaces · 2025Article
- GAPDH suppresses adenovirus-induced oxidative stress and enables a superfast production of recombinant adenovirus.Genes & diseases · 2024Article
- Evaluation of the Increased Genetic Resolution and Utility for Source Tracking of a Recently Developed Method for GenotypingMicroorganisms · 2024Article
- Adipose-derived mesenchymal stem cells (MSCs) are a superior cell source for bone tissue engineering.Bioactive materials · 2024Article
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- Advanced breast cancer diagnosis: Multiplex RT-qPCR for precise typing and angiogenesis profiling.Biochemistry and biophysics reports · 2024Article
- Effect of antibiotic monensin on cell proliferation and IGF1R signaling pathway in human colorectal cancer cells.Annals of medicine · 2023Article
- Designing and validation of specific primers for the quantitative detection of bacteria in sugarcane inoculant.Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] · 2023Article
- Integrated Transcriptome Analysis Identified Key Expansin Genes Associated with Wheat Cell Wall, Grain Weight and Yield.Plants (Basel, Switzerland) · 2023Article
22 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
22 authors at 8 institutions in 2 countries.
Funding
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.