title: 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.
authors.affiliation: Department of Neurology, and the Ministry of Education Key Laboratory of Child Development and Disorders, The Children's Hospital of Chongqing Medical University, Chongqing 400046, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Department of Cell Biology, the Third Military Medical University, Chongqing 400038, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Deng
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Zhengjian
authors.initials: Z
authors.lastname: Yan
authors.affiliation: Department of Biostatistics and Computational Biology, University of Rochester, Rochester, NY 14642, United States of America.
authors.forename: Yinglin
authors.initials: Y
authors.lastname: Xia
authors.affiliation: Department of Neurology, and the Ministry of Education Key Laboratory of Child Development and Disorders, The Children's Hospital of Chongqing Medical University, Chongqing 400046, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America.
authors.forename: Zhongliang
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; School of Bioengineering, Chongqing University, Chongqing 400044, China.
authors.forename: Jixing
authors.initials: J
authors.lastname: Ye
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Youlin
authors.initials: Y
authors.lastname: Deng
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Departments of General Surgery and Neurology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
authors.forename: Zhonglin
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Min
authors.initials: M
authors.lastname: Qiao
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Departments of General Surgery and Neurology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
authors.forename: Ruifang
authors.initials: R
authors.lastname: Li
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America.
authors.forename: Sahitya K
authors.initials: SK
authors.lastname: Denduluri
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wei
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Department of Orthopaedic Surgery, the Second Affiliated Hospital of Lanzhou University, Lanzhou 730000, China.
authors.forename: Lianggong
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Department of Orthopaedic Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250012, China.
authors.forename: Shun
authors.initials: S
authors.lastname: Lu
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Department of Surgery, West China Hospital of Sichuan University, Chengdu 610041, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Departments of General Surgery and Neurology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
authors.forename: Shengli
authors.initials: S
authors.lastname: Tang
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America; Ministry of Education Key Laboratory of Diagnostic Medicine, and the Affiliated Hospitals of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America.
authors.forename: Hue H
authors.initials: HH
authors.lastname: Luu
authors.affiliation: Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America.
authors.forename: Rex C
authors.initials: RC
authors.lastname: Haydon
authors.affiliation: Department of Neurology, and the Ministry of Education Key Laboratory of Child Development and Disorders, The Children's Hospital of Chongqing Medical University, Chongqing 400046, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, United States of America.
authors.forename: Tong-Chuan
authors.initials: TC
authors.lastname: He
authors.affiliation: Department of Neurology, and the Ministry of Education Key Laboratory of Child Development and Disorders, The Children's Hospital of Chongqing Medical University, Chongqing 400046, China.
authors.forename: Li
authors.initials: L
authors.lastname: Jiang
mesh_terms: D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005456:Fluorescent Dyes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012269:Ribosomal Proteins / Q000235:genetics; D015854:Up-Regulation; D060509:Wnt3A Protein / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012269:Ribosomal Proteins; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007041:Hypoxanthine Phosphoribosyltransferase