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Quantitative PCR Analysis of Double-Stranded RNA-Mediated Gene Silencing in Fungi

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Book cover Laboratory Protocols in Fungal Biology

Part of the book series: Fungal Biology ((FUNGBIO))

Abstract

Gene silencing in fungi produces a range of phenotypes based on the different amounts of target mRNA that are degraded by the RNAi machinery in each transformed strain. Detection of this range of variation when analyzing groups of transformants requires a fast and sensitive method. Quantitative or real-time PCR of reverse-transcribed target mRNA is particularly well suited for this analysis.

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Notes

  1. 1.

    Check www.gene-quantification.info for examples and download links.

  2. 2.

    For example Invitrogen Trizol Reagent. Cat. Number: 15596-026. http://products.invitrogen.com/ivgn/product/15596026.

  3. 3.

    Optional: the addition of formaldehyde denatures the high secondary structure of the RNA molecule for a clear visualization.

  4. 4.

    Roche cat. Numbers: 05081955001/05091284001/05081963001. http://www.roche-applied-science.com/proddata/gpip/3_6_8_39_1_3.html.

  5. 5.

    Invitrogen cat. Numbers: 18080-093/18080-044/18080-085. http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Amplification -and-Expression-Profiling/Reverse-Transcription-and-cDNA-Synthesis/RT___cDNA_Synthesis-Misc/SuperScript.html.

  6. 6.

    For Roche Lightcycler 480 there is an import tool available in http://www.hartfaalcentrum.nl/index.php?main=files&sub=0

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Authors and Affiliations

  1. Department of Microbiologia y Genetica-CIALE, Universidad de Salamanca, C/Río Duero, 12, Campus de Villamayor, Villamayor, Salamanca, 37185, Spain

    José J. de Vega-Bartol, Vega Tello, Jonathan Niño, Virginia Casado & José M. Díaz-Mínguez

Authors
  1. José J. de Vega-Bartol
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  2. Vega Tello
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  3. Jonathan Niño
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  4. Virginia Casado
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  5. José M. Díaz-Mínguez
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Corresponding author

Correspondence to José M. Díaz-Mínguez .

Editor information

Editors and Affiliations

  1. , Department of Biochemistry, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Vijai Kumar Gupta

  2. , Department of Biochemistry, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Maria G. Tuohy

  3. , Department of Biochemistry, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Manimaran Ayyachamy

  4. , Manufacturing Sciences and Technology, Pfizer Ireland Pharmaceuticals, The Pfizer Biotech Campus at Grange Castle, Clondalkin, N/A, Dublin, Ireland

    Kevin M. Turner

  5. , School of Natural Sciences, National University of Ireland Galway, University Road, Galway, N/A, Ireland

    Anthonia O’Donovan

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de Vega-Bartol, J.J., Tello, V., Niño, J., Casado, V., Díaz-Mínguez, J.M. (2013). Quantitative PCR Analysis of Double-Stranded RNA-Mediated Gene Silencing in Fungi. In: Gupta, V., Tuohy, M., Ayyachamy, M., Turner, K., O’Donovan, A. (eds) Laboratory Protocols in Fungal Biology. Fungal Biology. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-2356-0_22

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