TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR.
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 plat...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4501803?pdf=render |
| _version_ | 1819226031219802112 |
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| author | Qian Zhang Jing Wang Fang Deng Zhengjian Yan Yinglin Xia Zhongliang Wang Jixing Ye Youlin Deng Zhonglin Zhang Min Qiao Ruifang Li Sahitya K Denduluri Qiang Wei Lianggong Zhao Shun Lu Xin Wang Shengli Tang Hao Liu Hue H Luu Rex C Haydon Tong-Chuan He Li Jiang |
| author_facet | Qian Zhang Jing Wang Fang Deng Zhengjian Yan Yinglin Xia Zhongliang Wang Jixing Ye Youlin Deng Zhonglin Zhang Min Qiao Ruifang Li Sahitya K Denduluri Qiang Wei Lianggong Zhao Shun Lu Xin Wang Shengli Tang Hao Liu Hue H Luu Rex C Haydon Tong-Chuan He Li Jiang |
| author_sort | Qian Zhang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-23T10:19:01Z |
| format | Article |
| id | doaj.art-a5cc3100ace8469397c2c5569935d97c |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-23T10:19:01Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-a5cc3100ace8469397c2c5569935d97c2022-12-21T17:50:44ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01107e013266610.1371/journal.pone.0132666TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR.Qian ZhangJing WangFang DengZhengjian YanYinglin XiaZhongliang WangJixing YeYoulin DengZhonglin ZhangMin QiaoRuifang LiSahitya K DenduluriQiang WeiLianggong ZhaoShun LuXin WangShengli TangHao LiuHue H LuuRex C HaydonTong-Chuan HeLi JiangThe 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.http://europepmc.org/articles/PMC4501803?pdf=render |
| spellingShingle | Qian Zhang Jing Wang Fang Deng Zhengjian Yan Yinglin Xia Zhongliang Wang Jixing Ye Youlin Deng Zhonglin Zhang Min Qiao Ruifang Li Sahitya K Denduluri Qiang Wei Lianggong Zhao Shun Lu Xin Wang Shengli Tang Hao Liu Hue H Luu Rex C Haydon Tong-Chuan He Li Jiang TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR. PLoS ONE |
| title | TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR. |
| title_full | TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR. |
| title_fullStr | TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR. |
| title_full_unstemmed | TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR. |
| title_short | TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR. |
| title_sort | tqpcr a touchdown qpcr assay with significantly improved detection sensitivity and amplification efficiency of sybr green qpcr |
| url | http://europepmc.org/articles/PMC4501803?pdf=render |
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