|
| Status |
Public on Jul 07, 2010 |
| Title |
Cells + DOPA 24 h rep 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Fungal cells, no DOPA, 24 h
|
| Organism |
Candida albicans |
| Characteristics |
reference: pooled control samples
strain: SC5314
|
| Treatment protocol |
The cells were flash frozen in liquid nitrogen and RNA was extracted as described by Hayes et al (Methods 2002;26:281-290)
|
| Growth protocol |
C. albicans strain SC5314 was grown in the dark for 72 h at 35 °C in cultures with and without addition of L-DOPA to 1 mM. Samples were taken at 6, 24, 48 and 72 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted as described by Hayes et al (Methods 2002;26:281-290). cDNA was prepared from the RNA samples by reverse transcription. In brief, 2mg of oligo (dT)12-18 primer (500mg/mL)(Promega) was added to 50mg of RNA and made up to 10mL with RNase-free H2O, incubated for 10 min at 70 °C and snap-cooled on ice for 1 min.
|
| Label |
Cy3
|
| Label protocol |
To each control RNA sample, 15mL of labelling master mix (1x RT buffer,1 mM DTT, 500 mM dATP, 500 mM dCTP, 500 mM dGTP,100 mM dTTP) was added followed by 3 mL of Cy3-dUTP (Amersham Biosciences). The triplicate samples from cells grown in L-DOPA were individually labelled with Cy5-dUTP (Amersham Biosciences) by the same method. Following the addition of the label, 2 mL of Superscript II reverse transcriptase (Invitrogen) was added, mixed thoroughly and incubated at 42 °C for2 h. The reaction was stopped by addition of 1.5 mL of 20 mM EDTA, followed by 1.5 mL of 500 mM NaOH and incubated for 10 min at 70 °C to degrade the RNA. This reaction was neutralized by addition of 1.5 mL of 500 mM HCl. Following reverse transcription, the samples were mixed and purified in a GFX purification column (Amersham Biosciences) following the manufacturer’s procedure, then concentrated in a Speed Vac to 20 mL.
|
| |
|
| Channel 2 |
| Source name |
Fungal cells, plus DOPA, 24 h
|
| Organism |
Candida albicans |
| Characteristics |
strain: SC5314
agent: 1 mM L-DOPADOPA
time point: 24 h
|
| Treatment protocol |
The cells were flash frozen in liquid nitrogen and RNA was extracted as described by Hayes et al (Methods 2002;26:281-290)
|
| Growth protocol |
C. albicans strain SC5314 was grown in the dark for 72 h at 35 °C in cultures with and without addition of L-DOPA to 1 mM. Samples were taken at 6, 24, 48 and 72 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted as described by Hayes et al (Methods 2002;26:281-290). cDNA was prepared from the RNA samples by reverse transcription. In brief, 2mg of oligo (dT)12-18 primer (500mg/mL)(Promega) was added to 50mg of RNA and made up to 10mL with RNase-free H2O, incubated for 10 min at 70 °C and snap-cooled on ice for 1 min.
|
| Label |
Cy5
|
| Label protocol |
To each control RNA sample, 15mL of labelling master mix (1x RT buffer,1 mM DTT, 500 mM dATP, 500 mM dCTP, 500 mM dGTP,100 mM dTTP) was added followed by 3 mL of Cy3-dUTP (Amersham Biosciences). The triplicate samples from cells grown in L-DOPA were individually labelled with Cy5-dUTP (Amersham Biosciences) by the same method. Following the addition of the label, 2 mL of Superscript II reverse transcriptase (Invitrogen) was added, mixed thoroughly and incubated at 42 °C for2 h. The reaction was stopped by addition of 1.5 mL of 20 mM EDTA, followed by 1.5 mL of 500 mM NaOH and incubated for 10 min at 70 °C to degrade the RNA. This reaction was neutralized by addition of 1.5 mL of 500 mM HCl. Following reverse transcription, the samples were mixed and purified in a GFX purification column (Amersham Biosciences) following the manufacturer’s procedure, then concentrated in a Speed Vac to 20 mL.
|
| |
|
| |
| Hybridization protocol |
For each of the four time points, four independent hybridizations were done for labelled RNA from L-DOPA-grown cells versus the labelled, pooled control samples. Samples were pre-hybridized in 5x SSC/1% SDS/1% BSA for 45 min at 42 °C, washed five times in water, once in isopropanol and air-dried. The labelled samples were added to 20 mL of hybridization solution (50% formamide/10x SSC/0.2% SDS), boiled to denature for 3 min and carefully added to the printed part of the microarray slide and the lifter slip placed on the slide. The slides were incubated at 42 °C overnight. Slides were washed at room temperature in 2x SSC/1% SDS for 15 min, 1x SSC/0.2% SDS for 8 min and 0.1x SSC/0.2% SDS for 5 min. The slides were dried by centrifugation.
|
| Scan protocol |
The slides were scanned at different wavelengths so that when the dual images were opened together, each spot lit up either as a yellow colour or in varying shades of green or red, depending on the relative level of expression of the individual gene in each spot. Signals on the slides were located with a ScanArray 4000 Microarray Analysis System, and quantified with QuantArray AcquisitionSoftware.
|
| Description |
Cells + DOPA 24 h rep 2
|
| Data processing |
The raw data were normalized according to the Lowess algorithm, using the program GeneSpring to generate ratio data for further analysis. For each set of quadruplicate data, the median ratio of experimental/control was calculated. A cutoff of 2-fold or greater ratio change indicating up- or down-regulation of a gene was regarded as significant.
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| |
|
| Submission date |
Feb 16, 2010 |
| Last update date |
Jul 07, 2010 |
| Contact name |
Frank C Odds |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Aberdeen
|
| Department |
School of Medical Sciences
|
| Lab |
AFG
|
| Street address |
Institute of Medical Sciences
|
| City |
Aberdeen |
| ZIP/Postal code |
AB25 2ZD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL7476 |
| Series (1) |
| GSE20338 |
Candida albicans DOPA-grown cells |
|
