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Sample GSM509598 Query DataSets for GSM509598
Status Public on Jul 07, 2010
Title Cells + DOPA 24 h rep 4
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 4
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.
 
Submission date Feb 16, 2010
Last update date Jul 07, 2010
Contact name Frank C Odds
E-mail(s) f.odds@abdn.ac.uk
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

Data table header descriptions
ID_REF
VALUE Lowess normalized log2 ratios, experimental vs pooled controls.

Data table
ID_REF VALUE
CA00114_01 0.064265367
CA00127_01 0.223497432
CA00132_01 0.088063285
CA00133_01 -0.154327544
CA00144_01 -0.175078865
CA00152_01 0.173448437
CA00157_01 0.297129774
CA00170_01 0.544027727
CA00218_01 0.39196036
CA00223_01 0.369904851
CA00241_01 0.096122705
CA00250_01 0.223803346
CA00251_01 -0.166119776
CA00283_01 0.207499211
CA00288_01 -0.90794496
CA00295_01 0.126643853
CA00329_01 -0.189237749
CA00330_01 0.179416778
CA00331_01 -0.031889484
CA00332_01 0.210949623

Total number of rows: 6266

Table truncated, full table size 142 Kbytes.




Supplementary file Size Download File type/resource
GSM509598.txt.gz 591.5 Kb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data included within Sample table

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