|
| Status |
Public on Feb 15, 2016 |
| Title |
THP-1_UT_8h_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
THP-1_Untreated_8h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: THP-1 monocytic cell line
protocol: differentiated with PMA
treatment: control
time: 8 h
|
| Treatment protocol |
Nearly, 8 x 106 THP-1 cells were seeded in T-75 cm2 flasks in RMPI with 10% FBS. The cells were then differentiated with 30 nM PMA for 16-18 h. Following differentiation, the cells were allowed resting for 2-3 h. The cells were then treated with 0.1 mM vitamin C (vitC) and subsequently RNA was isolated from untreated control flasks and vit C treated flasks as 8 h, 24 h, 48 h and 96 h post vit C-addition.
|
| Growth protocol |
THP-1 cells were maintained in RPMI 1640 in 10% FBS. The cells were passaged at nearly 80% confluency. The experiment was performed at ~99% cell viability as determined by trypan blue exclusion method.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At respective time-point, the media was removed from the control and vit C-treated flasks. This was immediately followed by addition of TRI reagent and swirling the flasks in order to lyse the adhered cells. The cell lysates were centrifuged at 5000 rpm for 15 min at 4C. The supernatant was used for RNA extraction using bromochloropropane method.Briefly, 1/10th volume of bromochloropropane (Molecular Research Center, USA) was added to the samples and vigorously shaken for 10 sec. The samples were then incubated for 10 min at room temperature (for phase separation). The aqueous phase was separated by centrifuging the samples at 12000 rpm for 15 min at 4°C. The RNA in the aqueous phase was precipitated with 1/100th volume of polyacryl carrier and isopropanol (0.6 volumes, for 30 min), centrifuged (12,000 rpm, 4°C for 15 min) and washed with 75% ethanol and then air-dried. Total RNA was dissolved in 100 μl of DEPC-treated water.
|
| Label |
Cy3
|
| Label protocol |
The samples were labeled using Agilent Quick Amp Kit (Part number: 5190-0442). 500ng of total RNA was reverse transcribed using oligodT primer tagged to T7 promoter sequence. cDNA thus obtained was converted to double stranded cDNA in the same reaction. Further the cDNA was converted to cRNA in the in-vitro transcription step using T7 RNA polymerase enzyme and Cy3 dye was added into the reaction mix. During cRNA synthesis Cy3 dye was incorporated into the newly synthesized strands. cRNA obtained was cleaned up using Qiagen RNeasy columns (Qiagen, Cat No: 74106).
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| |
|
| Hybridization protocol |
600ng of labeled cRNA were hybridized on Custom Human Whole Genome 8x60K Array 8x60k designed by Genotypic Technology Private Limited (AMADID: 027114)using the Gene Expression Hybridization kit (Part Number 5190-0404; Agilent) in Sure hybridization Chambers (Agilent) at 65º C for 16 hours. Hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327).
|
| Scan protocol |
Scanned using the Agilent Microarray G2505C scanner (Agilent Technologies)
|
| Description |
Gene expression of PMA-differentiated untreated-THP-1 cells at 8 h
UT_8h_1
|
| Data processing |
Images were quantified using Feature Extraction Software ( Agilent). Feature extracted raw data was analyzed using GeneSpring GX Version software from Agilent. Normalization of the data was done in GeneSpring GX using the 75th percentile shift
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| |
|
| Submission date |
Sep 24, 2015 |
| Last update date |
Feb 15, 2016 |
| Contact name |
Genotypic technology |
| E-mail(s) |
[email protected]
|
| 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 |
GPL13252 |
| Series (1) |
| GSE73421 |
Gene expression profiling of THP-1 cells on treatment with vitamin C |
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