Selection and Evaluation of Reference Genes for Expression Studies with Quantitative PCR in the Model Fungus Neurospora crassa under Different Environmental Conditions in Continuous Culture

Abstract

Neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. However, in order to optimize N. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. Reverse-transcription quantitative PCR (RT-qPCR) is a technique that provides a high-throughput platform from which to measure the expression of a large set of genes over time. The selection of a suitable reference gene is critical for gene expression studies using relative quantification, as this strategy is based on normalization of target gene expression to a reference gene whose expression is stable under the experimental conditions. This study evaluated twelve candidate reference genes for use with N. crassa when grown in continuous culture bioreactors under different light and temperature conditions. Based on combined stability values from NormFinder and Best Keeper software packages, the following are the most appropriate reference genes under conditions of: (1) light/dark cycling: btl, asl, and vma1; (2) all-dark growth: btl, tbp, vma1, and vma2; (3) temperature flux: btl, vma1, act, and asl; (4) all conditions combined: vma1, vma2, tbp, and btl. Since N. crassa exists as different cell types (uni- or multi-nucleated), expression changes in a subset of the candidate genes was further assessed using absolute quantification. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes serve as a reliable reflection of transcript, and not gene copy number, fluctuations. The results of this study identified genes that are appropriate for use as reference genes in RT-qPCR studies with N. crassa and demonstrated that even with the presence of different cell types, relative quantification is an acceptable method for measuring gene expression changes during growth in bioreactors.

Suppressing the Neurospora crassa circadian clock while maintaining light responsiveness in continuous stirred tank reactors

“…The three significant differences between the experimental setup and results generated for this manuscript compared to the results presented by Crosthwaite and coworkers was the use of dynamic agitation to disperse the cellular material through the experiment, a significantly slower dilution rate (60.3 ± 0.7 mL/h vs. 0.074 ± 0.004 mL/h) to maintain steady state, and choice of reference genes ( btl and vma2 ) 32 . The amount of CO 2 generated remained steady between 3500–4000 ppm for both LD and DD experiments and confirmed that a steady metabolic state was maintained during these experiments ( Fig.…”

“…Periodic patterns in frq transcription are considered a reliable indicator of circadian gene oscillations in N. crassa 9 . These patterns were determined by RT-qPCR using stable reference genes for continuous culture conditions ( btl and vma2 ) 32 . The periodicity of this overt rhythm can be entrained by exposing the cultures to light.…”

“…To account for changes in multi-nucleated cellular states and to validate that RT-qPCR data was determining transcriptional changes and not changes in gene copy numbers our group has published data involving several reference genes using samples generated from these CSTRs 32 . Plotting both the RNA: DNA ratio and C T values for four genes under DD and LD conditions over time indicated that C T changes were due to fluctuations in transcript copy number.…”

“…The second issue in processing the samples from this batch control experiment was instability of the reference genes validated from the continuous culture experiments used for RT-qPCR. In the DD and LD experiments presented in this manuscript, a detailed study had previously been published which addressed the issue of reference gene stability under the continuous culture conditions used for these experiments 32 . In that publication, btl and vma2 were identified as stable reference genes for cultivating N. crassa in continuous culture bioreactors with dynamic agitation.…”

Identification of suitable reference genes during the formation of chlamydospores in Clonostachys rosea 67‐1

“…β ‐tubulin was used to normalize N‐acety1‐β‐D‐glucosaminidase gene in C. rosea when confronting with Fusarium culmorum (Mamarabadi, Jensen, & Lübeck, ), however, it was not available in mycoparasitic process (Sun, Li, & Sun, ; Sun, Sun, & Li, ). In Neurospora crassa , the expression of act was not affected by temperature, but it was inhibited under all‐dark conditions (Cusick et al., ). Therefore, selection of suitable reference genes under a specific condition is crucial for accurate gene normalization and function certification.…”