|
| Status |
Public on Jan 16, 2014 |
| Title |
doxy treated exp 6 |
| Sample type |
RNA |
| |
|
| Source name |
transfected HCT-116 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT-116
treatment: dox-induced DN-Tcf4 expression to repress Wnt signaling
agent: mock-treated
|
| Treatment protocol |
treatment was for 17.5 hr +/- 4 ug/ml doxycycline and/or 5 mM sodium butyrate
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions. RNA was purified using Ribopure (Ambion) RNA isolation kit. Total RNA samples were quantitated by UV spectrophotometry (OD260/280). Quality of the total RNA was assessed by using and Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA).
|
| Label |
Alexa-555
|
| Label protocol |
First and second strand cDNA was prepared from the total RNA samples. cRNA target was prepared from the DNA template and verified on the Bioanalyzer.
|
| |
|
| Hybridization protocol |
cRNA was fragmented to uniform size and hybridized to Agilent Human v2 Whole Genome 4x44K arrays (Design ID 026652).
|
| Scan protocol |
Slides were washed and scanned on the Agilent G2565 Microarray Scanner.
|
| Description |
Gene expression
Doxy Exp 6
|
| Data processing |
Agilent Feature Extraction software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies, Santa Clara, CA).
Analytical approach:
The overall analytical approach was to use human whole genome microarray analyses to first identify the identity of all genes whose expression was up- or downregulated by butyrate, in a statistically significant manner, by two-fold or more, in the absence of doxycycline induction of DN-Tcf4. We then repeated the analysis in the presence of doxycycline, and considered those genes whose expression was modulated by butyrate in the absence of doxycycline (fully intact Wnt signaling) but not similarly modulated in the presence of doxycycline (repressed Wnt signaling).
Thus, we were interested in identifying those genes that are differentially expressed (> two-fold, P< 0.01) in the NaB vs. Ctrl comparison but not differentially expressed (> two-fold, P< 0.01) in the Doxy + NaB vs. Doxy comparison. The NaB vs., Ctrl comparison yields all genes differentially expressed after exposure to 5 mM butyrate, while the Doxy + NaB vs. Doxy comparison yields all butyrate-modulated genes except those that are Wnt targets, since the doxycycline induction combined with butyrate treatment efficiently results in DN-Tcf4 expression, which inhibits Wnt signaling and Wnt-dependent gene expression.
Analyzed_data_Differentially_Expressed_Genes.xls is linked as a supplementary file on the Series record.
|
| |
|
| Submission date |
Jan 16, 2014 |
| Last update date |
Jan 16, 2014 |
| Contact name |
Michael Bordonaro |
| E-mail(s) |
mbordonaro@tcmedc.org
|
| Phone |
570-504-9646
|
| Organization name |
The Commonwealth Medical College
|
| Department |
Basic Sciences
|
| Lab |
Bordonaro
|
| Street address |
525 Pine Street
|
| City |
Scranton |
| State/province |
PA |
| ZIP/Postal code |
18509 |
| Country |
USA |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE54127 |
Butyrate Induced Changes in Wnt Signaling-Specific Gene Expression in Colorectal Cancer Cells |
|
