Selection of reference genes for quantitative real-time RT-PCR assays in different morphological forms of dimorphic zygomycetous fungus Benjaminiella poitrasii

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.

Fig 1. Specificity of primers used for RT-qPCR analysis in B. poitrasii.

A) Agarose gel electrophoresis and analysis of amplified products obtained from RT-qPCR. The electrophoresis was done on 2% agarose gel. The names of candidate reference genes are mentioned on top. The figures on left indicate the size of PCR amplicons in base pairs. B) Melting curves indicating single peaks for three representative genes. Arrow indicates the melting curve for the RT-qPCR reaction without template.

Fig 2. Distribution overview of expression of each candidate reference gene in absolute Ct values for RNA samples of different morphological forms in B. poitrasii.

Samples 1, yeast cells grown in YPG (1%) medium for 12 h; Sample 2, yeast cells grown in YPG medium for 24h; Sample 3, yeast cells grown in YPG (0.1%) medium for 12 h; Sample 4, hyphae grown in YP medium for 12 h; Sample 5, hyphae grown in YP medium for 24 h; Sample 6, hyphae grown in YPG(0.1%) for 12 h.; Sample 7, 5 d old sporangiospores; Sample 8, 10 d old sporangiospores; Sample 9, 15 d old sporangiospores; Sample 10, 7 d old zygospores; Sample 11, 15 d old zygospores; Sample 12, 30 d old zygospores.

Fig 3. geNorm—based ranking of the candidate reference genes during different morphological stages of B. poitrasii.

Genes were ranked from the least stable (on the left) to the most stable (on the right) according to their M value (Y axis). This classification was independently performed by using different sets of conditions. Vegetative stage, 6 samples of yeast and hyphal cells; Asexual stage, 3 samples of sporangiospores; Sexual stage, 3 samples of zygospores as described in Fig 2 legend.

Fig 4. Pairwise variation analysis between NFn and NFn+1 to determine the optimal number of genes for reliable normalization.

Values below the 0.15 threshold mean that n genes might be sufficient.

Fig 5. Quantification of ornithine decarboxylase (Bpodc) gene transcripts by RT-qPCR in different morphological forms of B. poitrasii.

The amount of Bpodc transcripts present in different morphological forms of B. poitrasii (Y, yeast cells; H, hyphae; S, sporangiospores; Z, zygospores) at different time points (h, hours; d, days) was measured and normalized with the transcripts from Ubc and WS-21 (A) and Tub-b (B) as reference.