|
| Status |
Public on Dec 04, 2008 |
| Title |
HtTA-LMX1B_Expt1_d0 |
| Sample type |
RNA |
| |
|
| Source name |
HtTA-LMX1B cells grown in the presence of doxycycline (non-induced cells)
|
| Organism |
Homo sapiens |
| Characteristics |
Tet-off inducible HeLa cell line (HtTA) expressing human LMX1B protein upon doxycycline removal
|
| Biomaterial provider |
Juergen Prestel and Ralph Witzgall
|
| Treatment protocol |
HtTA-LMX1B cells were grown in Dulbecco’s modified Eagle’s medium (DMEM high glucose with L-Glutamine) containing 10% FBS, 200 µg/ml Geneticin, 30 ng/ml doxycycline, and 300 µg/ml Hygromycin B. Medium was changed every 2 days. The presence of doxycycline prevents expression of human LMX1B. These cells thus represent the non-induced reference sample for the microarray analysis.
|
| Growth protocol |
HtTA-LMX1B cells were grown in Dulbecco’s modified Eagle’s medium (DMEM high glucose with L-Glutamine) containing 10% FBS, 200 µg/ml Geneticin, 30 ng/ml doxycycline, and 300 µg/ml Hygromycin B. Medium was changed every 2 days. The presence of doxycycline prevents expression of human LMX1B. These cells thus represent the non-induced reference sample for the microarray analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were isolated using the Nucleospin RNA II kit (Macherey-Nagel), including an on-column DNase I treatment to eliminate genomic DNA contamination, following the manufacturer's recommendations.
|
| Label |
Biotin
|
| Label protocol |
Sample preparation was carried out in accordance with the Affymetrix GeneChipTM Whole Transcript (WT) Sense Target Labeling Assay Manual (Rev. 2). Three-hundred nanograms of DNase-treated total RNA were used to generate Biotin-Labeled sense strand (ss) DNA.
|
| |
|
| Hybridization protocol |
Following fragmentation and terminal labeling, ssDNA products (5.5 µg) were hybridized to the array for 16 h at 45 °C at 60 rpm in a rotating chamber. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450.
|
| Scan protocol |
Fluorescent signals were measured with an Affymetrix GeneChipTM Scanner 3000-7G. Signal intensity calculation was performed using the RMA algorithm as implemented in the Affymetrix Expression Console 1.1 software.
|
| Description |
Reference sample for the microarray analysis (non-expressing cells), replicate 1.
|
| Data processing |
Significance analysis was performed with ArrayAssist Software (Agilent). Transcripts showing a fold change above 2-fold and a p-value below 0.05 were considered as significantly regulated.
|
| |
|
| Submission date |
Jul 04, 2008 |
| Last update date |
Dec 04, 2008 |
| Contact name |
Anne Rascle |
| E-mail(s) |
anne.rascle@vkl.uni-regensburg.de
|
| Phone |
+49 (0)941 2806598
|
| Organization name |
University of Regensburg
|
| Street address |
Universitaetsstrasse
|
| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE12008 |
Identification of LMX1B target genes in a tet-off inducible HeLa cell line and in the mouse kidney |
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