Reference Gene Selection for Quantitative Real-Time PCR of Mycelia from under High-Temperature Stress.

Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes, and , were tested for their adequacy in (). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia of , which were treated with high-temperature stress at 37°C for 0, 4, 8, 12, 18, and 24 hours. After treatment, expression stability of candidate genes was evaluated using three statistical software programs: geNorm, NormFinder, and BestKeeper. According to geNorm, had the lowest M values in strains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 was (0.030), and the best candidate reference gene in strain 18N44 was (0.047). In BestKeeper analysis, the standard deviation (SD) values of , , , , , , and in strain 18 and those of and in strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, only and were found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses, was the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction in mycelia under high-temperature stress.