|
| Status |
Public on Mar 11, 2013 |
| Title |
CgPDR1OE - repeat 1 - mAdbID:56651 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cg84 Control_Cy3
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
strain: Cg84
genotype/variation: wild type
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Sample Extraction Protocol
Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA) and the RNeasy MiniElute cleanup kit (Qiagen, Valencia, CA).
|
| Label |
cy3
|
| Label protocol |
Cy3 Sample Labeling Protocol
Total RNA was reverse transcribed to cDNA to incorporate the fluorescent Cy3-dUTP (GE Health Care, Piscataway, NJ).
|
| |
|
| Channel 2 |
| Source name |
CgPDR1OE_Cy5
|
| Organism |
Nakaseomyces glabratus |
| Characteristics |
strain background: Cg84u (uracil auxotroph of Cg84)
strain: CgPDR1OE
genotype/variation: CgPDR1 overexpression
|
| Treatment protocol |
To create pGRB2.2-CgPDR1, the PDR1 gene from Candida glabrata strain 13928 was amplified by PCR with primers PDR2 and PDR4. The Cg13928 PDR1 ORF contains a gain of function mutation that results in constitutively high expression of CDR1. The PCR product was blunt ended with T4 polymerase and ligated into the pCgACU vector, which had been previously digested with Sma1. The pCgACU-CgPDR1 plasmid was transformed into 84u.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Sample Extraction Protocol
Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA) and the RNeasy MiniElute cleanup kit (Qiagen, Valencia, CA).
|
| Label |
cy5
|
| Label protocol |
Cy5 Sample Labeling Protocol
Total RNA was reverse transcribed to cDNA to incorporate the fluorescent Cy5-dUTP (GE Health Care, Piscataway, NJ).
|
| |
|
| |
| Hybridization protocol |
Hybridization Protocol
The microarrays were prehybridized at 42°C in prehybridization buffer (5× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 1% bovine serum albumin [BSA], 0.1% SDS) for 30 to 60 min and then hybridized to the labeled cDNA in 50 ul of hybridization buffer (25% formamide, 5× SSC, 0.2% SDS, 20 ug/ml poly[dA]40-60, 200 ug/ml Cot-1 DNA [Invitrogen], 80 ug/ml yeast tRNA) overnight at 42°C. The microarrays were washed three times in wash buffer A (1× SSC, 0.05% SDS) and washing buffer B (0.1× SSC).
|
| Scan protocol |
Creator: GenePix Pro 5.1.0.4
Scanner: GenePix 4000B [82425]
ScanPower: 100;; 100
LaserPower: 3.16;; 3.1
Temperature: 29.81
|
| Description |
mAdb experiment ID: 56651
|
| Data processing |
CgPDR1OE Data Processing Protocol
Calculation Method: Default Ratio: ChanB/ChanA (Cy5/Cy3); Signal calculation: Median Intensity minus Median Background; Any Features designated Control were excluded; Normalization method: 50th Percentile (Median) using all spot filtered Genes; Spot Filter Options: Include Spots not flagged BAD or Not Found AND Target diameter >= 0 um AND Target diameter <= 300 um AND Both Chan A and Chan B Percentage of Target Pixels Saturated <= 50% AND Both Chan A and Chan B Signal/Background Ratios >= 2.000 AND Both Chan A and Chan B Signal >= 500 Override other Chan A & B criteria and Include if Chan A Signal >= 5000 OR Chan B Signal >= 5000 Signal Values were floored at 100 (ChanA and ChanB); Data was extracted and aligned by the Inventory Well ID; Within array multiple occurrences of Well ID were reduced to a single instance by selecting the one with the strongest signal (Chan A + Chan B); Note: For all GenePix results from Axon scanned arrays Chan A is CY3 and Chan B is CY5.
|
| |
|
| Submission date |
Apr 05, 2012 |
| Last update date |
Mar 11, 2013 |
| Contact name |
Jason A Noble |
| E-mail(s) |
noblej2@mail.nih.gov
|
| Phone |
301-4968956
|
| Organization name |
National Institutes of Health
|
| Department |
NIAID
|
| Lab |
Clinical Mycology
|
| Street address |
10 Center Drive, Building 10, Room 11N228
|
| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL8174 |
| Series (1) |
| GSE37071 |
The Candida glabrata transcription factor Stb5p is a negative regulator of ATP-binding cassette transporters |
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