Abstract
Benjaminiella poitrasii, a dimorphic non-pathogenic zygomycetous fungus, exhibits a morphological yeast (Y) to hypha (H) reversible transition in the vegetative phase, sporangiospores (S) in the asexual phase and zygospores (Z) in the sexual phase. To study the gene expression across these diverse morphological forms, suitable reference genes are required. In the present study, 13 genes viz. ACT, 18S rRNA, eEF1α, eEF-Tu,eIF-1A, Tub-α, Tub-b, Ubc, GAPDH, Try, WS-21, NADGDH and NADPGDH were evaluated for their potential as a reference, particularly for studying gene expression during the Y-H reversible transition and also for other asexual and sexual life stages of B. poitrasii. Analysis of RT-qPCR data using geNorm, normFinder and BestKeeper software revealed that genes such as Ubc, 18S rRNA and WS-21 were expressed at constant levels in each given subset of RNA samples from all the morphological phases of B. poitrasii. Therefore, these reference genes can be used to elucidate the role of morpho-genes in B. poitrasii. Further, use of the two most stably expressed genes (Ubc and WS-21) to normalize the expression of the ornithine decarboxylase gene (Bpodc) in different morphological forms of B. poitrasii, generated more reliable results, indicating that our selection of reference genes was appropriate.
Highlights
Fungi display morphological plasticity and differentiate in to vegetative, asexual and sexual forms during their life cycle
Selection of B. poitrasii morphological forms of different ages for RTqPCR studies based on intracellular protein, cAMP and NAD(P)H contents
As the differences in the concentrations of protein, cAMP and NAD(P)H were significant in different morphological forms of different ages, the corresponding samples were used for the analysis of candidate reference genes (S1 Fig)
Summary
Fungi display morphological plasticity and differentiate in to vegetative, asexual and sexual forms during their life cycle. RNA quantification is an important tool being used to study the correlation between gene expression and morphological outcome of an organism. Reverse transcriptase PCR (RT-PCR), cDNA microarray and quantitative real-time RT-PCR (RT-qPCR) are commonly used for RNA quantification [1]. Among these methods, RT-qPCR is preferred due to its high sensitivity, robustness, high specificity, good reproducibility and a wide range [2, 3]. To get the unbiased results in RT-qPCR, gene expression data must be normalized with appropriate reference gene(s). The number of genes involved in basic cellular functions, such as ribosomal genes
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