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Quantitative analysis of multiple genes’ expressions based on a novel competitive RT-PCR assay

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Abstract

We established a novel gene expression analysis platform, Multiplex Competitive RT-PCR Using Fluorescent Universal Primers (MCF-PCR), to study multi-gene expression patterns simultaneously. This platform combines fluorescent universal primers, multiplex competitive RT-PCR, and capillary electrophoretic separation, which ensures MCF-PCR a reliable, medium-throughput, cost-effective technology for gene expression profiling. With cloned standard DNAs, the detection limits, precision, and sensitivity of MCF-PCR were evaluated and compared with that of the assay without adding competitive templates and real-time PCR, respectively. The results showed that detection limit was 3.125 × 103 to 3.2 × 106 copies, and 10 % copy differences between two samples can be detected by MCF-PCR. To validate MCF-PCR, we analyzed expression profile of five genes in interleukin (IL)-4/IL-13 pathway in peripheral blood of 20 healthy adults and 20 allergic dermatitis patients; three genes including IL-4, IL-13, and STAT6 were found differentially expressed in the two sample groups, which maybe key players in IL-4/IL-13 immunological signaling pathway and need further function analysis.

Principle of MCF-PCR. cDNA was amplified using chimeric primers, each containing 18-20 nt target-complementary sequence (solid blue/black) and 18 nt universal primer-complementary sequence (solid red). Subsequent PCR amplifications using universal primers to yield fluorescent-labeled amplification products.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (no. 81273276, 81072448, 30972824, and 30671939), Science and Technology Commission of Shanghai Yangtze River Delta Technology joint research project (no. 10495810200), and Basic Research Project of Shanghai Science and Technology Commission (no. 11JC1410300). We would like to thank Shanghai Biowing Applied Biotechnology Co., Ltd. for providing sequencing service.

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Authors and Affiliations

  1. Department of Laboratory Medicine, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China

    Jia Li, Li-hui Lin, Juan Wang, Xia Peng, Ya-nan Liu & Li Li

  2. Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, China

    Jun-hua Xiao & Yu-xun Zhou

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  1. Jia Li
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  2. Li-hui Lin
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  3. Juan Wang
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  4. Xia Peng
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  5. Ya-nan Liu
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  6. Jun-hua Xiao
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  7. Yu-xun Zhou
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  8. Li Li
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Correspondence to Yu-xun Zhou or Li Li.

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Li, J., Lin, Lh., Wang, J. et al. Quantitative analysis of multiple genes’ expressions based on a novel competitive RT-PCR assay. Anal Bioanal Chem 405, 1353–1360 (2013). https://doi.org/10.1007/s00216-012-6518-1

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  • DOI: https://doi.org/10.1007/s00216-012-6518-1

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