|
| Status |
Public on May 09, 2018 |
| Title |
THP-1_PBS_6h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
THP-1-PBS, 6h, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: THP-1 macrophages
timepoint: 6 h
infection: uninfected
|
| Treatment protocol |
PMA-differentiated THP-1 cells were infected with Candida glabrata strains
|
| Growth protocol |
THP-1 monocytes were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a humified incubator with 5% CO2. For THP-1 cells differentiation, cells were treated with 16 nM PMA for 12 h and then replenished with fresh RPMI medium. Yeast cells were grown in YPD medium at 30°C for 14-16 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed using the Trizol method. as per manufacturer’s protocol. The concentration and purity of the RNA extracted were evaluated using the Nanodrop Spectrophotometer (Thermo Scientific; 1000).
|
| Label |
Cy3
|
| Label protocol |
Samples for Gene expression were labeled using the Agilent Quick-Amp labeling Kit (p/n5190-0442). 500 ng each of total RNA were reverse transcribed at 40°C using oligo dT primer tagged to a T7 polymerase promoter and converted to double stranded cDNA. Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up using Qiagen RNeasyMini kit columns (Qiagen, Cat No: 74106) and quality assessed for yields and specific activity using the Nanodrop ND-1000.
|
| |
|
| Hybridization protocol |
600 ng of labeled cRNA sample were fragmented at 60ºC and hybridized on to a Agilent designed Human Gene expression Microarray 8x60K (AMADID No: G4858A_39494) arrays. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of (Agilent Technologies, In situ Hybridization kit, Part Number 5190-0404). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours.
|
| Scan protocol |
Agilent Microarray Scanner (Agilent Technologies, Part Number G2600D).
|
| Description |
Gene expression after 6 h in uninfected THP-1 cells
|
| Data processing |
Images were quantified using the Feature Extraction Software ( Agilent). Feature extracted raw data was analyzed using GeneSpring GX software from Agilent. Normalization of the data was done in GeneSpring GX using the quantile normalization method .
|
| |
|
| Submission date |
Aug 29, 2016 |
| Last update date |
May 09, 2018 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL21061 |
| Series (1) |
| GSE86176 |
Expression of genes in THP-1 macrophages in response to C. glabrata wt, Cgvps34Δ and Cgyps(1-11)Δ cells. |
|
