African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12487

Full Length Research Paper

Assessment and validation of reference genes for qRT-PCR normalization in local cowpea (Vigna unguiculata L.) varieties in response to sterilized and unsterilized soil conditions

Frank Kwarteng Amoako
  • Frank Kwarteng Amoako
  • Institute of Plant Nutrition and Soil Science, Kiel University, Hermann-Rodewald-Strasse 2, 24118 Kiel, Germany.
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Francis Anti Amoako
  • Francis Anti Amoako
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Owuraku Amponsah Abu
  • Owuraku Amponsah Abu
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Melchizedek Asamoah Amponsah
  • Melchizedek Asamoah Amponsah
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Emmanuel Digooh
  • Emmanuel Digooh
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Lily Naa Adoley Batsa
  • Lily Naa Adoley Batsa
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Agnes Nimo Bosompem
  • Agnes Nimo Bosompem
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Clara Ama Adutwumwaa
  • Clara Ama Adutwumwaa
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Belinda Owiah Baiden
  • Belinda Owiah Baiden
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Felicity Animah Anarfi
  • Felicity Animah Anarfi
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Jeffrey Kankam Boateng
  • Jeffrey Kankam Boateng
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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Michael Ackah
  • Michael Ackah
  • School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, People’s Republic of China.
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Aikins Nyamekye
  • Aikins Nyamekye
  • BB 64, Adisababa Street, Offinso-Ashanti, Ghana.
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Obed Adjei
  • Obed Adjei
  • Department of Horticulture, Faculty of Agriculture, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi-Ashanti, Ghana.
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Ruth Prempeh
  • Ruth Prempeh
  • Council for Scientific and Industrial Research-Crops Research Institute (CSIR-CRI), Fumesua, P. O. Box 3785, Kumasi, Ghana.
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  •  Received: 26 February 2024
  •  Accepted: 05 June 2024
  •  Published: 30 June 2024

Abstract

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely employed as a mandatory analysis in biological work to analyze and validate gene expression; however, its precision is greatly hampered by the robustness of the internal control genes employed for normalization. In this study, we selected suitable housekeeping genes in leaves of cowpea for normalization of expression analysis when exclusively exposed to sterilized and unsterilized soil conditions. To validate appropriate internal control genes for qRT-PCR for three cowpea varieties grown under sterilized and unsterilized soil conditions, we investigated the expression stability of five conventional reference genes in cowpea leaves using four approaches: BestKeeper, Delta Ct, geNorm and NormFinder, incorporated in the RefFinder web-based software. The four algorithms employed revealed that Actin is the reference gene with the highest precision when used as an internal control, with EF1-α and 18S rRNA being classified as optimal housekeeping genes for local cowpea under both soil conditions. However, GAPDH and β-tubulin exhibited highly unstable expression patterns in cowpea under both soil conditions based on the standardized algorithms. These analyzed genes shed light on the molecular physiology of cowpea plants in response to varied soil conditions and suggested standardized reference genes for laboratories.

 

Key words: Vigna unguiculata, reference gene, qRT-PCR, unsterilized condition, nodule.