|
| Status |
Public on Mar 02, 2013 |
| Title |
non-targeting siRNA control, no doxorubicin, rep3 |
| Sample type |
RNA |
| |
|
| Source name |
HCT116 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116 cells
sirna: non-targeting siRNA control
treatment: no doxorubicin
|
| Treatment protocol |
Cells were transfected with control or TAF3 siRNA. 48 hours after siRNA transfection, the cells were treated with 0.5 μM doxorubicin for 24 hours or left untreated.
|
| Growth protocol |
HCT116 cells were cultured on tissue culture plates in the Dulbecco's Modified Eagle Medium (DMEM, Gibco)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted using the Rneasy kit (Qiagen)
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared with the MessageAmp Premier RNA Amplification kit (Applied Biosystems, Foster City, CA). Briefly, 200 ng of total RNA was used to synthesize the first strand of cDNA using ArrayScript reverse transcriptase and an oligo(dT) primer bearing a T7 promoter. The single-stranded cDNA was then converted into a double-stranded DNA (dsDNA) by DNA polymerase I in the presence of E. coli RNase H and DNA ligase. The dsDNA was then served as a template for in vitro transcription in a reaction containing biotin-labeled UTP, unlabeled NTPs and T7 RNA Polymerase. The amplified, biotin-labeled antisense RNA (aRNA) was purified and the quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano kit.
|
| |
|
| Hybridization protocol |
Standard Illumina hybridization protocol. 750 ng of aRNA in 5 ul was mixed with 10 ul of hybridization reagents and heated at 65C for 5 min. After cooled to room temperature, total 15 ul of the hybridization solution was applied to Illumina HumanHT-12 v3 chip. The chip was incubated for about 18 hours at 58C. Then washed and stained with streptavidin-Cy3.
|
| Scan protocol |
Standard Illumina scanning protocol usig Illumina BeadArray Reader. The scanning was done using standard DirectHyb Gene Expression protocol with the following settings: Factor=1, PMT=587, Filter=100%.
|
| Description |
non-targeting siRNA control, no doxorubicin
Illumina HumanHT-12 v3 Beadchip
|
| Data processing |
Raw data was extracted using Illumina BeadStudio software without normalization. Results were analyzed by GeneSpring GX 10.0.2 using simple median-scaling. Genes were filtered on Flags Present or Marginal.
|
| |
|
| Submission date |
Jan 16, 2013 |
| Last update date |
Mar 02, 2013 |
| Contact name |
Xiaolin Wu |
| E-mail(s) |
forestwu@mail.nih.gov
|
| Phone |
301-846-7677
|
| Organization name |
Frederick National Laboratory for Cancer Research/SAIC-Frederick Inc
|
| Department |
Cancer Research Technology Program
|
| Lab |
Genomics Laboratory
|
| Street address |
8560 Progress Drive, C3001
|
| City |
Frederick |
| State/province |
MD |
| ZIP/Postal code |
21701 |
| Country |
USA |
| |
|
| Platform ID |
GPL6947 |
| Series (2) |
| GSE43541 |
H3K4me3 Interactions with TAF3 Regulate Preinitiation Complex Assembly and Selective Gene Activation [Illumina BeadArray] |
| GSE43542 |
H3K4me3 Interactions with TAF3 Regulate Preinitiation Complex Assembly and Selective Gene Activation |
|
