Allison Cockrell - 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

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        Kathleen D. Cusick, Lisa A. Fitzgerald, Russell K. Pirlo,   Allison L. Cockrell, Emily R. Petersen, and   Justin C. Biffinger (2014) PLoS   One 9(12): e112706




          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.    


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