Abstract
Volvariella volvacea (V. volvacea), commonly referred to as Chinese (paddy straw) mushroom, is a basidiomycete with a protein-rich volva and pileus. Selecting appropriate reference genes is a crucial step in the normalization of quantitative real-time PCR data. Therefore, 12 candidate reference genes were selected from the V. volvacea transcriptome based on previous studies and then BestKeeper, geNorm, and NormFinder were used to identify reference genes stably expressed during different developmental stages and conditions. Of the 12 candidate reference genes, SPRY domain protein (SPRYp), alpha-tubulin (TUBα), cyclophilin (CYP), L-asparaginase (L-asp), and MSF1-domain-containing protein (MSF1) were the most stably expressed under different experimental conditions, while 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), and beta-actin (ACTB) were the least stably expressed. This investigation not only revealed potential factors influencing the suitability of reference genes, but also identified optimal reference genes from a pool of candidate genes under a wide range of conditions.
Highlights
Quantitative real-time PCR (RT-qPCR) has emerged as a powerful and popular tool used for rapid and accurate assessment of changes in gene expression [1,2,3]
The most commonly used method is RT-qPCR, where a suitable reference gene is necessary for normalization of results
12 candidate reference genes in V. volvacea were investigated to determine the most stably expressed under different conditions
Summary
Quantitative real-time PCR (RT-qPCR) has emerged as a powerful and popular tool used for rapid and accurate assessment of changes in gene expression [1,2,3]. When normalizing target gene expression, selecting a stable reference gene is extremely important, especially for samples with fluctuating expression levels [7, 8]. The use of unsuitable reference genes in RT-qPCR analysis has yielded unreliable and confusing results [9, 10]. Certain reports have supported merging expression of at least three reference genes when normalizing RT-qPCR results [11, 12]. The same reference genes often cannot be used for different tissues and cells, even when the samples are derived from the same species. Selection of suitable reference genes based on a given experimental design or species is necessary [13, 14]
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