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Stepwise Optimization of Real-Time RT-PCR Analysis

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Plant Genome Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2653))

Abstract

Quantitative real-time reverse transcription PCR (qRT-PCR) analysis has been used routinely to quantify gene expression levels. Primer design and the optimization of qRT-PCR parameters are critical for the accuracy and reproducibility of qRT-PCR analysis. Computational tool-assisted primer design often overlooks the presence of homologous sequences of the gene of interest and the sequence similarities between homologous genes in a plant genome. This sometimes results in skipping the optimization of qRT-PCR parameters due to the false confidence in the quality of the designed primers. Here we present a stepwise optimization protocol for single nucleotide polymorphisms (SNPs)-based sequence-specific primer design and sequential optimization of primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference and target gene. The goal of this optimization protocol is to achieve a standard cDNA concentration curve with an R2 ≥ 0.9999 and efficiency (E) = 100 ± 5% for the best primer pair of each gene, which serves as the prerequisite for using the 2−ΔΔCT method for data analysis.

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Acknowledgments

The authors thank the USDA National Institute of Food and Agriculture Hatch project 02685 and North Carolina State University for the startup funds to the Liu laboratory.

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Authors and Affiliations

  1. Department of Horticultural Science, North Carolina State University, Raleigh, NC, USA

    Nathan A. Maren, James R. Duduit, Debao Huang, Fanghou Zhao & Wusheng Liu

  2. Mountain Crop Improvement Lab, Department of Horticultural Science, Mountain Horticultural Crops Research and Extension Center, North Carolina State University, Mills River, NC, USA

    Nathan A. Maren & Thomas G. Ranney

  3. National Center for Soybean Improvement, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China

    Fanghou Zhao

Authors
  1. Nathan A. Maren
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  2. James R. Duduit
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  3. Debao Huang
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  4. Fanghou Zhao
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  5. Thomas G. Ranney
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  6. Wusheng Liu
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Corresponding author

Correspondence to Wusheng Liu .

Editor information

Editors and Affiliations

  1. Division of Plant Sciences, University of Missouri, Columbia, MO, USA

    Bing Yang

  2. Department of Crop Genetics, John Innes Centre, Norwich, UK

    Wendy Harwood

  3. Syngenta Crop Protection, LLC., Research Triangle Park, NC, USA

    Qiudeng Que

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Maren, N.A., Duduit, J.R., Huang, D., Zhao, F., Ranney, T.G., Liu, W. (2023). Stepwise Optimization of Real-Time RT-PCR Analysis. In: Yang, B., Harwood, W., Que, Q. (eds) Plant Genome Engineering. Methods in Molecular Biology, vol 2653. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3131-7_20

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  • DOI: https://doi.org/10.1007/978-1-0716-3131-7_20

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3130-0

  • Online ISBN: 978-1-0716-3131-7

  • eBook Packages: Springer Protocols

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