|
| Status |
Public on May 25, 2007 |
| Title |
ED14-MMH_Prox1_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MMH-pCDNA3.1
|
| Organism |
Mus musculus |
| Characteristics |
Geneticin regulated met-murine-hepatocyte cell line treated with an empty vector.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
- RNA was isolated with RNeasy kit (Qiagen) according to the manufactures standard protocol
- Subsequent DNase I treatment
|
| Label |
Cy3
|
| Label protocol |
- totalRNA was transcribed, amplified and labeled according to BD Atlas SMART fluorescent probe amplification kit with following modifications:
- RNA template was hydrolized after transcription (high pH, 30 min/70 degree celcius)
- PCR amplification progress was monitored and stopped before reaching saturation
|
| |
|
| Channel 2 |
| Source name |
MMH-Prox1
|
| Organism |
Mus musculus |
| Characteristics |
Geneticin regulated met-murine-hepatocyte cell line transfected with Prox1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
- RNA was isolated with RNeasy kit (Qiagen) according to the manufactures standard protocol
- Subsequent DNase I treatment
|
| Label |
Cy5
|
| Label protocol |
- totalRNA was transcribed, amplified and labeled according to BD Atlas SMART fluorescent probe amplification kit with following modifications:
- RNA template was hydrolized after transcription (high pH, 30 min/70 degree celcius)
- PCR amplification progress was monitored and stopped before reaching saturation
|
| |
|
| |
| Hybridization protocol |
- hybridization according to Agilent 60-mer oligo microarray processing protocol v2.1 with the following modifications:
2 micro gram labeled DNA per channel were mixed
no fractionation
probes were denatured for 3 min at 98 degree celcius
hybridization at 63 degree celcius
SSPE washes as recommended
slides were dipped in acetonitril (10 s) then dried
|
| Scan protocol |
- scanned with a G2505B Microarray Scanner (Agilent Technologies)
- quantification software: 'Automic Imageprocessing for Microarrays'
(for details: http://www.techfak.uni-bielefeld.de/ags/ai/projects/microarray/)
|
| Description |
- RNA quality control with Agilent Bioanalyzer 2100
- Dye incorporation with NanoDrop ND-1000
Keywords = Met murine hepatocytes - Hex - HNF4a - MMP-2 - Type-IV collagen
|
| Data processing |
- dye swap design
- Normalization of the log2(cy3/cy5)-ratios with pinwise non-linear loess regression
- afterwards we did a scaling procedure by division of each value with the standard deviation of the normalized log2-ratios (variance normalization)
- as statistical software we use 'R'
|
| |
|
| Submission date |
May 22, 2007 |
| Last update date |
May 30, 2007 |
| Contact name |
Gabriela Salinas |
| E-mail(s) |
Gabriela.Salinas-Riester@medizin.uni-goettingen.de
|
| Organization name |
Universitaetsmedizin Goettingen
|
| Department |
Department of Human Genetics
|
| Lab |
NGS Integrative Genomics
|
| Street address |
Justus-von-Liebig-Weg 11
|
| City |
Goettingen |
| State/province |
Lower-Saxony |
| ZIP/Postal code |
37077 |
| Country |
Germany |
| |
|
| Platform ID |
GPL2872 |
| Series (1) |
|
