The experiments were preformed in 500 mL flasks with 50 mL YPD medium. Cells grown to early-log phase (OD600=1.0) and then 0.02% (v/v) of D-limonene was added to the cultures, then the cells were incubated at 30°C for 2 h with constant shaking at 200 rpm. Cells were harvested at 2000g for 5 min, then washed twice with double distilled water and stored at -80°C until RNA preparation.
Growth protocol
A single colony of the strain was inoculated to a 250 mL flask with 25 mL YPD media (10 g/L yeast extract, 20 g/L tryptone and 20 g/L glucose) and cultured 24 h at 200 rpm and 30°C as seed for the following culture. The seeds from pre-culture were inoculated into flask with an initial cell density of OD660=0.01. The growth of yeast was monitored by optical density at 600 nm (OD600) with a spectrophotometer. Incubation condition was 30°C with constant shaking at 200 rpm. All experiments were performed in triplicates.
Extracted molecule
total RNA
Extraction protocol
Total RNA of either control or D-limonene-treated cells was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions, including a DNase digestion step. The RNA was quantified and checked in the Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 260 and 280 nm. Integrity of isolated RNA was verified by automated electrophoresis system (Bio-Rad, Hercules, CA, USA).
Label
Cy3
Label protocol
Sample labeling and hybridization reactions were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Briefly, for each sample 1 μg of total RNA was linearly amplified and labeled with Cy3-dCTP. Labelled cRNAs purified with Qiagen RNAeasy Mini Kit, and sample concentration and specific activity (pmol Cy3/μg cRNA) were measured in a NanoDrop® ND-1000 spectrophotometer.
Hybridization protocol
A total of 1μg of labeled cRNA was prepared for fragmentation adding 11 μl 10 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, heated at 60 °C for 30 min, and finally diluted by addition with 55μl 2×GE Hybridization buffer. A volume of 100 μl of hybridization solution was then dispensed in the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent Hybridization Oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
The hybridized arrays were washed, fixed and scanned using the Agilent DNA microarray scanner (part number G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%). following Agilent Technologies’ guidelines.
Description
control
Data processing
The resulting text files extracted from Agilent Feature Extraction Software (version 10.5.1.1) were imported into the Agilent GeneSpring GX software (version 11.0) for further analysis. The microarray data sets were normalized in GeneSpring GX using the Agilent FE one-color scenario (mainly median normalization), The positive effect of this median normalization is illustrated in Box-plot, and genes marked present or marginal in all samples (“All Targets Value”) were chosen for data analysis. Differentially expressed genes were identified through Fold-change screening.
