Computational protocol: Effect of textile dyes on activity and differential regulation of laccase genes from Pleurotus ostreatus grown in submerged fermentation

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[…] Total RNA was isolated from frozen mycelia harvested at different fermentation times, using TRIZOL (Invitrogen) extraction, and was spectrophotometrically quantified by determining the absorbance ratio at OD260/280. RNA was treated with RNAse-free DNase I (Invitrogen). The final RNA concentration was set to 500 ng/µl. Subsequently, 1 µg of total RNA was reverse-transcribed into cDNA in a 20 µl volume using the SuperScript™II Reverse Transcriptase (Invitrogen) by following the manufacturer protocol.The procedure for reverse transcription quantitative PCR experiments was adapted from (Castanera et al. ). RT-qPCRs were performed in a StepOnePlus® (Applied Biosystems), using SYBR green dye to detect product amplification. A set of specific primers was designed for the amplification of the transcript from the four laccase genes identified in the genome (Table ). Primers corresponding to the panel of reference genes were designed using the filtered model transcript sequence of PC15 (v2.0) (http://www.jgi.doe.gov) and the Express Primer Express® 3.0 (Applied Biosystems) (Additional file : Table S1). With a final volume of 20 µl, each reaction mixture contained 10 µl Maxima Probe/ROX qPCR Master Mix (2X) (ThermoScientific), 200 nM forward and reverse primer, and a 1 µl 1:10 dilution of the RT product. Amplifications were performed with an initial 5 min step of 95 °C followed by 40 denaturation cycles at 95 °C for 30 s and primer annealing and extension at 60 °C for 40 s. The melting curves ranged from 60 to 95 °C and temperature was increased in increments of 0.3 °C. StepOne software was used to confirm the occurrence of specific amplification peaks. All RT-qPCR reaction were carried out in triplicate with template-free negative control being performed in parallel. The crossing-point (Cp) values and relative fluorescence units were recorded, with the latter used to calculate amplification efficiencies via linear regression. The PCR efficiency (E) and the regression coefficient (R2) were calculated using the slope of the standard curve according to the equation E = [10−(1/slope)−1] × 100 %. [...] Four genes of different functional class were selected as reference candidates. The gene panel used in this study contained housekeeping genes, such as glyceraldehyde 3-phosphate dehydrogenase (gpd), β-tubulin (tub), actin (act) and peptidase (pep) (Additional file : Table S1). The expression of the genes was evaluated in six samples corresponding to our experimental conditions. GeNorm (Vandesompele et al. ) and NormFinder (Andersen et al. ) algorithms were applied to rank the four candidates according to their expression stability, and a reference index consisting of the geometric mean of the best-performing candidates was used for RT-qPCR data normalization.Data pre-processing was performed using Microsoft Excel 2007 and included efficiencies and reference gene normalization. The fold expression was calculated by the 2−Δ ΔCt method as described by (Pfaffl ) (Eq. ).1ratio =(Etarget)ΔCptarget(control-sample)(Eref)ΔCpref(control-sample) In the above equation Etarget is the real-time PCR efficiency of target gene transcript: Eref is the real-time PCR efficiency of a reference gene transcript; ΔCptarget is the CP deviation of control-sample of the target gene transcript. All other multiples comparisons were performed using the statistical analysis software SAS 2002 by SAS Institute Inc., Cary, NC, USA. […]

Pipeline specifications

Software tools Primer Express, NormFinder
Application qPCR
Organisms Pleurotus ostreatus