|
| Status |
Public on Dec 01, 2011 |
| Title |
5-AzaC 5uM TSA 0.3 uM 2 |
| Sample type |
RNA |
| |
|
| Source name |
Prostate cancer LNCaP cells
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: 5-AzaC 5uM TSA 0.3 uM
experiment: 2
cell line: LNCaP
|
| Treatment protocol |
Cells were treated with dimethylsulfoxide (DMSO) as a control and 5-AzaC (5 microM), and/or TSA (0.3 microM). For combined treatments, TSA was added after 12 hr of pre-treatment with 5-AzaC or control. After 36 hr, all cell treatments were harvested for RNA extraction. DMSO, 5-AzaC and TSA n=5; TSA/5-AzaC n=4 independent experiments.
|
| Growth protocol |
LNCaP cells were plated at 1x10^6 cells per 10 cm2 for 48 hours post treatment
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with TRIZOL reagent according to the manufacturer protocol
|
| Label |
Biotin
|
| Label protocol |
Refer to Liu et al (PNAS 101: 9740-9744 (2004)). 5 ug of total RNA was labeled in reverse transcription at 37C for 90 mins by using biotin-labeled rand-octomer oligo primer. The RT reaction mix was further denatured by using 0.5 N NaOH / 1 mM EDTA at 65C for 15 mins and neutralized by 1 M Tris HCl pH 7.6. Biotin signal was detected with an Alexa 647-Streptavidin conjugate.
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| |
|
| Hybridization protocol |
The chips were hybridized on Tecan HS 4800 hybridization station. Chips were pre-hybridized at 25C for 30 min in the buffer of 6x SSPE/ 30% formamide/ 1x Denhardt's solution. The chips were further hybridized with labeled target in 6x SSPE/ 30% formamide at 25C for 18 hr. Hybridization, post-hybridization washing in 0.75x TNT (Tris, sodium, Tween 20) (see PNAS) at 37C for 40 mins. The chips were stained by streptavidin-alexa647 (1:500) dilution in TNT for 30 mins. Post-staining washing in 1x TNT FOR 40 min. Rinse with water and spin dry.
|
| Scan protocol |
Arrays were scanned on the GenePix scanner and analyzed using the GenePix Pro software package.
|
| Data processing |
Raw gpr data files were MAS.5 median-based normalization and filtered for probes with low intensities (<10), minimum fold-change (i.e., less than 20% of expression data values with at least a 1.5-fold change in either direction from the gene’s median value) and >50% missing data were excluded. Class comparisons between treatments were carried out using randomized block univariate t-tests across independent experiments.
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| |
|
| Submission date |
Aug 23, 2011 |
| Last update date |
Dec 01, 2011 |
| Contact name |
Robert Scott Hudson |
| Organization name |
NIH
|
| Department |
NCI
|
| Lab |
The Laboratory of Human Carcinogenesis
|
| Street address |
National Cancer Institute Room Blgd.37/3044
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20852 |
| Country |
USA |
| |
|
| Platform ID |
GPL14184 |
| Series (1) |
| GSE31620 |
MicroRNA-1 is a candidate tumor suppressor and prognostic marker |
|
