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.