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

doi:10.1371/journal.pone.0132666

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	TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR
	Zhang, Q; Wang, J; Deng, F; Yan, ZJ; Xia, YL; Wang, ZL; Ye, JX; Deng, YL; Zhang, ZL; Qiao, M
刊名	PLOS ONE
	2015-07-14
卷号	10 期号:7
ISSN号	1932-6203
DOI	10.1371/journal.pone.0132666
文献子类	Article
英文摘要	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.
学科主题	Science & Technology - Other Topics
出版地	SAN FRANCISCO
资助项目	国家自然科学基金项目 ; 美国国立卫生研究院项目
项目编号	National Institutes of Health [AT004418, AR50142, AR054381] ; National Natural Science Foundation of China (NSFC Grant) [81371452, 81271770] ; University of Chicago Core Facility grant from the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health [UL1 TR000430]
语种	英语
WOS记录号	WOS:000358194900073
资助机构	NSFC ; NIH
内容类型	期刊论文
源URL	[http://ir.lzu.edu.cn/handle/262010/179377]
专题	第二临床医学院_期刊论文
通讯作者	He, TC (reprint author), Chongqing Med Univ, Childrens Hosp, Dept Neurol, Chongqing 400046, Peoples R China.
推荐引用方式 GB/T 7714	Zhang, Q,Wang, J,Deng, F,et al. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR[J]. PLOS ONE,2015,10(7).
APA	Zhang, Q.,Wang, J.,Deng, F.,Yan, ZJ.,Xia, YL.,...&He, TC .(2015).TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR.PLOS ONE,10(7).
MLA	Zhang, Q,et al."TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR".PLOS ONE 10.7(2015).