The cell was removed to 50 mL conical tubes and centrifuged (3,220 g, 4˚C) to discard the supernatant completely; the cell was stored at -80˚C prior to RNA extraction.
Growth protocol
Wild type (SC5314) and rhb1∆/rhb1∆ (CCT-D1) Candida albicans strains were subcultured in 40 mL of fresh SD (synthetic defined) medium (250mL flask) for 5.5 hours at 30˚C and 220rpm to reach O.D. 600 nm approximately at 4.0.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted by RNeasy® kit (74106, QIAGNE, Valencia, CA) following by manufacturer’s manual. Extracted total RNA was subsequently treated with Turbo DNA-free™ DNase (Ambion, Austin, TX) for 1 h at 37˚C to degrade residual DNA. The quality and quantity of total RNA are determined using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) or agarose gel electrophoresis and ND-1000 spectrophotometer (NanoDrop Technologies).
Label
Cy5
Label protocol
cDNA synthesis, aRNA synthesis and Cy-dye coupling was carried out by using Amino Allyl MessageAmp™ II aRNA Amplification Kit (AM1753, Ambion) following by manufacturer’s manual. Briefly, the first strand of cDNA was then prepared from total RNA using ArrayScript™ reverse transcriptase. After processing, DNA polymerase and RNase H was further used for second strand cDNA synthesis. In vitro transcription by using T7 RNA polymerase and purified cDNA as the template, the amino allyl UTP (aaUTP) incorporated aRNA (amino allyl-modified aRNA) was synthesized. aRNA was purified and performed Cy5 (Amersham Cy5 Mono-Reactive Dye Pack, PA25001, GE Healthcare) or Cy3 (Amersham Cy3 Mono-Reactive Dye Pack, PA23001, GE Healthcare) dye coupling. Cy dye coupled aRNA is purified by ultrafiltration using RNeasy® MinElute™ Cleanup kit (QIAGEN).
Channel 2
Source name
Mid log-phase rhb1 mutant cultured in SD medium at 30˚C
The cell was removed to 50 mL conical tubes and centrifuged (3,220 g, 4˚C) to discard the supernatant completely; the cell was stored at -80˚C prior to RNA extraction.
Growth protocol
Wild type (SC5314) and rhb1∆/rhb1∆ (CCT-D1) Candida albicans strains were subcultured in 40 mL of fresh SD (synthetic defined) medium (250mL flask) for 5.5 hours at 30˚C and 220rpm to reach O.D. 600 nm approximately at 4.0.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted by RNeasy® kit (74106, QIAGNE, Valencia, CA) following by manufacturer’s manual. Extracted total RNA was subsequently treated with Turbo DNA-free™ DNase (Ambion, Austin, TX) for 1 h at 37˚C to degrade residual DNA. The quality and quantity of total RNA are determined using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) or agarose gel electrophoresis and ND-1000 spectrophotometer (NanoDrop Technologies).
Label
Cy3
Label protocol
cDNA synthesis, aRNA synthesis and Cy-dye coupling was carried out by using Amino Allyl MessageAmp™ II aRNA Amplification Kit (AM1753, Ambion) following by manufacturer’s manual. Briefly, the first strand of cDNA was then prepared from total RNA using ArrayScript™ reverse transcriptase. After processing, DNA polymerase and RNase H was further used for second strand cDNA synthesis. In vitro transcription by using T7 RNA polymerase and purified cDNA as the template, the amino allyl UTP (aaUTP) incorporated aRNA (amino allyl-modified aRNA) was synthesized. aRNA was purified and performed Cy5 (Amersham Cy5 Mono-Reactive Dye Pack, PA25001, GE Healthcare) or Cy3 (Amersham Cy3 Mono-Reactive Dye Pack, PA23001, GE Healthcare) dye coupling. Cy dye coupled aRNA is purified by ultrafiltration using RNeasy® MinElute™ Cleanup kit (QIAGEN).
Hybridization protocol
Pre-hybridized with 1% BSA 5x SSC and 0.1% SDS at 42 °C for 1hr. Subsequently washed with 0.1x SSC two times vigorously for 5 min at 25 °C. Then washed in ddH2O for 30 sec at 25 °C, and rinsed with isopropanol prior to drying and hybridizing. The array was hybridized with Cy-3 and Cy-5 labeled aRNA (+ 20µg of poly A and yeast tRNA) in Corning Microarray Hybridization Chamber (No.2551, Corning). The hybridization was reacted at 65˚C water bath for 16 hours. Array then washed in 2x SSC (+ 0.03 % SDS) at 65 °C for 2min. Sub-washed in 2x SSC, 1x SSC, 0.2x SSC and 0.1x SSC, each at 25 °C for 2min prior to drying and scanning.
Scan protocol
The slide were scanned using Axon GenePix 4000B scanner (Axon Instruments, Inc., Union City, CA); Genepix software was used to pick up the spot.
Data processing
Lowess normalization was used to remove intensity-based variation across channels. The following linear model was then fitted for each of the spot on DNA microarrays: y ijkl = strain i + dye j + array k + ε ijkl, where strain i stands for the ith strain for i=1, 2, dye j stands for the jth dye effect for j=1,2, array k stands for the overall array effect and is a random effect, and ε ijkl is the residual term for random variation that is not caught by the above model. y ijkl is the intensity we want to model. The genes with significant strain effects are selected using False Discovery Rate (FDR)=0.05, which is equivalent to p-value ≤ 0.0148. The analysis was carried out with JMP Genomics (SAS Institute Inc., Cary, NC) and the R package qvalue
