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

Abstract

Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes, TUB, TUA, GADPH, EF1, 18S, GTP, ACT, UBI, UBC, and H2A, were tested for their adequacy in Lentinula edodes (L. edodes). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia of L. edodes, 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, TUB had the lowest M values in L. edodes strains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 was TUB (0.030), and the best candidate reference gene in strain 18N44 was UBI (0.047). In BestKeeper analysis, the standard deviation (SD) values of UBC, TUA, H2A, EF1, ACT, 18S, and GTP in strain 18 and those of GADPH and GTP in strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, only UBI and TUB were found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses, TUB was the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction in L. edodes mycelia under high-temperature stress.

Figure 1

Analysis of 10 reference genes using geNorm. Low M values indicate high expressional stability. In iterative steps, genes with the lowest stability (i.e., the highest M value) are removed. When the threshold value of M is smaller than 1.5, the candidate reference parameters are applicable. The two less stable genes (UBC and GTP) are eliminated.

Figure 2

Gene expression stability of 10 reference genes ranked according to geNorm analysis. Gene expression studies for identification of most stable reference genes under high-temperature stress using geNorm software based on average expression stability value (M). The direction of arrow indicates the most and least stable reference genes in graphs. (a) strain 18; (b) strain 18N44.

Figure 3

Analysis of 10 reference genes SD values using BestKeeper. Reference genes with an SD below 1 are considered stably expressed, and a smaller SD indicates a more stable reference gene. The results showed that UBI and TUB are stable genes (SD < 1).

Figure 4

Pairwise variation (Vn/n+1) values calculated by geNorm. The cut-off value to determine the optimal number of reference genes for qRT-PCR normalization is 0.150. Vn/n+1 < 0.15 indicates that the inclusion of an additional reference gene is not required. (a) strain 18; (b) strain 18N44.

Figure 5

Validation of the selected reference genes. Relative expression of hsp100 and hyd1 in mycelia of L. edodes strains 18 (a; b) and 18N44 (c; d). In the strain 18, TUB and ACT were the most stable candidate reference genes, and UBC was the least stable candidate reference gene. In the strain 18N44, TUB and TUA were the most stable candidate reference genes, and GTP was the least stable candidate reference gene. Data are shown as mean ± standard deviation (n = 3).