Strain CD36 incubated on reconstituted human oral epithelium (RHE; Skinethic, France) for 30 min at 37C, 5% CO2.
Biomaterial provider
Sullivan et al (1995), Microbiology 141: pp 1507-1521
Treatment protocol
Fungal cells were incubated on RHE for 30 min. Two mL of a solution containing two parts (v/v) of RNAlater solution (Ambion) and one part of filter-sterilized 10% (w/v) saponin (Sigma-Aldrich) in PBS was added to a 4-cm^2 Candida–RHE culture. To dislodge fungal cells, membranes were rinsed 3-4 times with the RNAlater-saponin solution, and the cell suspension was transferred to a 50-ml screw-cap tube along with the cut filter membranes, and immediately frozen and stored at -20°C.
Growth protocol
Candida cells from -80°C stocks were streaked on YPD agar, and grown for 20 h at 37°C or 64 h at 22°C. About 5 fungal colonies were resuspended and washed thrice in phosphate-buffered saline (PBS) and used to inoculate 10 mL filter-sterilized YPD to a density of 2 × 10^5 cells/mL. Cells were grown for 16-20 h at 22-25°C with shaking (200 rpm) and used to inoculate 10 mL filter-sterilized YPD to a density of 4 × 10^6 cells/mL. Cells were grown for 14 h at 37°C with shaking (200 rpm), harvested by centrifugation, washed thrice in PBS, and adjusted to a density of 4 × 10^7 cells/mL in PBS. Reconstituted human oral epithelium (RHE) tissues (4 cm^2) were inoculated with 200 μl of Candida cell suspension in PBS, and incubated on maintenance medium (MCDB153) at 37°C, 5% CO2, and 100% humidity.
Extracted molecule
total RNA
Extraction protocol
Cell suspensions containing RHE membranes in RNAlater solution (Ambion) were thawed at room temperature, vortexed for 5-10 sec, and centrifuged at 3200g for 5 min. The membranes were removed with clean forceps, the tubes again centrifuged, and the supernatant removed by slow aspiration. The cell pellet was used in RNA extraction with the RNeasy Mini Kit (Qiagen). Cell disruption was performed with a Mikro-Dismembrator S system (Sartorius Stedim Biotech, Göttingen, Germany). The cell pellet was suspended in 300 μl of buffer RLT (Qiagen), and the suspension transferred to a Teflon vessel with a tungsten-carbide bead (7 mm), pre-chilled with liquid N2, and shaken for 2 min at 2000 rpm in the dismembrator. The tungsten bead was quickly removed, another 400 μl RLT buffer added, and the homogenate used in RNA extraction following the RNeasy manufacturer's protocol. Total RNA was taken up in nuclease-free water and DNase I treated with DNAfree Turbo Kit (Ambion), quantified and purity assessed by 260/280 spectrophotometer measurements, and integrity checked on 1.2% TBE gels. Absence of contaminating DNA after DNase treatment was checked by PCR using RNA samples as template.
Label
Cy3
Label protocol
The Amino Allyl MessageAmp II aRNA Amplification kit (Ambion) was used for RNA amplification and labelling, following the manufacturer’s protocol. One μg of total RNA was used for reverse transcription, and amino-allyl-labelled RNA (aaRNA) amplified by T7-based in vitro transcription. The aaRNA obtained was Cy labelled, using N-hydroxysuccinimide esters of Cy3 and Cy5 dyes (GE Healthcare, Bucks, UK) as per the manufacturer’s instructions. Labelling efficiency was routinely assessed by measuring spectrophotometric absorbance at 550 nm (Cy3), 650 nm (Cy5), and 260 nm (DNA).
Sullivan et al (1995), Microbiology 141: pp 1507-1521
Treatment protocol
Fungal cells were in stationary phase after growth in yeast-extract peptone broth for 14 h at 37°C with shaking (200 rpm).
Growth protocol
Candida cells from -80°C stocks were streaked on YPD agar, and grown for 20 h at 37°C or 64 h at 22°C. About 5 fungal colonies were resuspended and washed thrice in phosphate-buffered saline (PBS) and used to inoculate 10 mL filter-sterilized YPD to a density of 2 × 10^5 cells/mL. Cells were grown for 16-20 h at 22-25°C with shaking (200 rpm) and used to inoculate 10 mL filter-sterilized YPD to a density of 4 × 10^6 cells/mL. Cells were grown for 14 h at 37°C with shaking (200 rpm), harvested by centrifugation, washed thrice in PBS, and adjusted to a density of 4 × 10^7 cells/mL in PBS.
Extracted molecule
total RNA
Extraction protocol
Cell suspensions were centrifuged (6000g, 5 min) and the cell pellet was used in RNA extraction with the RNeasy Mini Kit (Qiagen). Cell disruption was performed with a Mikro-Dismembrator S system (Sartorius Stedim Biotech, Göttingen, Germany). The cell pellet was suspended in 300 μl of buffer RLT (Qiagen), and the suspension transferred to a Teflon vessel with a tungsten-carbide bead (7 mm), pre-chilled with liquid N2, and shaken for 2 min at 2000 rpm in the dismembrator. The tungsten bead was quickly removed, another 400 μl RLT buffer added, and the homogenate used in RNA extraction following the RNeasy manufacturer's protocol. Total RNA was taken up in nuclease-free water and DNase I treated with DNAfree Turbo Kit (Ambion), quantified and purity assessed by 260/280 spectrophotometer measurements, and integrity checked on 1.2% TBE gels. Absence of contaminating DNA after DNase treatment was checked by PCR using RNA samples as template.
Label
Cy5
Label protocol
The Amino Allyl MessageAmp II aRNA Amplification kit (Ambion) was used for RNA amplification and labelling, following the manufacturer’s protocol. One μg of total RNA was used for reverse transcription, and amino-allyl-labelled RNA (aaRNA) amplified by T7-based in vitro transcription. The aaRNA obtained was Cy labelled, using N-hydroxysuccinimide esters of Cy3 and Cy5 dyes (GE Healthcare, Bucks, UK) as per the manufacturer’s instructions. Labelling efficiency was routinely assessed by measuring spectrophotometric absorbance at 550 nm (Cy3), 650 nm (Cy5), and 260 nm (DNA).
Hybridization protocol
About 5 μl of Cy-labelled aaRNA (1 μg of each treatment) was incubated at 70°C for 5 min, chilled on ice for 1 min, added to 55 μl DIG EasyHyb solution (Roche Diagnostics), and immediately added to a microarray slide, placed in a hybridization chamber. The slide was covered with a HybriSlip (Schleicher & Schuell), and incubated stationary at 42°C for 16-18 h in a hybridization oven. Slides were washed in 50-ml washing solutions at room temperature. Washes were for 10 min with 1×SSC, 0.2% SDS; 10 min with 0.1×SSC, 0.2% SDS; and 5 min with 0.1×SSC. Slides were dipped in 0.1×SSC and in sterile MilliQ water, quickly dried by centrifugation at 500g, and scanned immediately.
Scan protocol
Microarray slides were scanned with a GenePix 4000B scanner (Axon Instruments, Sunnyvale, CA, USA) at a resolution of 10 μm, using the auto PMT setting. Fluorescent intensity data were extracted using GenePix Pro 6.1 software (Axon Instruments).
Description
Biological replicate 3 of 3.
Data processing
GenePix result (gpr) files were uploaded into ArrayPipe (http://www.pathogenomics.ca /arraypipe/). Data were background subtracted with the normexp algorithm and normalized by loess normalization on each subgrid. Log2-transformed ratios of experimental/control intensities were calculated for each detected feature.
