To validate the expression profiles of the assembled genes obtained through sequencing data analysis, quantitative real-time (RT-qPCR) was performed for selected genes. Genes associated with biomass degradation processes were selected and are shown in Table S1 , together with the primers and annealing temperatures.
Quantification of gene expression was performed by continuously monitoring SYBR Green fluorescence. The reactions were performed in triplicate in a total volume of 6.25 µl. Each reaction included 3.12 µl of SYBR Green Master Mix (Invitrogen, Carlsbad, CA), 1.0 µl of direct and reverse primers, 0.5 µL of cDNA and 1.6 µl of water. The reactions were assembled in 384-well plates. PCR amplification-based expression profiling of the selected genes was performed using a gene for squalene-epoxidase as endogenous control. Four genes were tested as endogenous control: genes for actin, beta-tubulin, glyceraldehyde 3-phosphate dehydrogenase, and squalene-epoxidase. The last one had the best performance in RT-qPCR analysis, remaining constant in all treatments. The enzyme squalene-epoxidase catalyses the conversion of squalene to 2,3-(S) oxidosqualene, which is an intermediate in the synthesis of the fungal cell membrane component ergosterol. RT-qPCR was conducted in an ABI PRISM 7500 HT (Applied Biosystems, Foster City, CA). Gene expression was calculated via the Delta-Delta cycle threshold method [47] (link). All statistical comparisons were done using Student's t test (P<0.05). The obtained RT-qPCR results were in agreement with the RNA expression analyses of the generated assemblies. The same expression profile was observed for the genes encoding GH16, GH10, CE5, and GH5.Figure 1 shows the expression of the selected genes.
Quantification of gene expression was performed by continuously monitoring SYBR Green fluorescence. The reactions were performed in triplicate in a total volume of 6.25 µl. Each reaction included 3.12 µl of SYBR Green Master Mix (Invitrogen, Carlsbad, CA), 1.0 µl of direct and reverse primers, 0.5 µL of cDNA and 1.6 µl of water. The reactions were assembled in 384-well plates. PCR amplification-based expression profiling of the selected genes was performed using a gene for squalene-epoxidase as endogenous control. Four genes were tested as endogenous control: genes for actin, beta-tubulin, glyceraldehyde 3-phosphate dehydrogenase, and squalene-epoxidase. The last one had the best performance in RT-qPCR analysis, remaining constant in all treatments. The enzyme squalene-epoxidase catalyses the conversion of squalene to 2,3-(S) oxidosqualene, which is an intermediate in the synthesis of the fungal cell membrane component ergosterol. RT-qPCR was conducted in an ABI PRISM 7500 HT (Applied Biosystems, Foster City, CA). Gene expression was calculated via the Delta-Delta cycle threshold method [47] (link). All statistical comparisons were done using Student's t test (P<0.05). The obtained RT-qPCR results were in agreement with the RNA expression analyses of the generated assemblies. The same expression profile was observed for the genes encoding GH16, GH10, CE5, and GH5.
Free full text: Click here
