|
| Status |
Public on Feb 27, 2007 |
| Title |
CT mouse hepatotoxicity 6F |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver
|
| Organism |
Mus musculus |
| Characteristics |
Swiss mice, Male, 10-12 weeks
|
| Biomaterial provider |
Central Drug Research Institute, India
|
| Treatment protocol |
Control mice
|
| Growth protocol |
Mice were kept under optimal conditions of light and temperature
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRI reagent (sigma)
|
| Label |
Cy3
|
| Label protocol |
CyScribe first strand cDNA labelling kit protocol (Amersham)
|
| |
|
| Channel 2 |
| Source name |
Liver, Drug treated
|
| Organism |
Mus musculus |
| Characteristics |
Swiss mice, Male, 10-12 weeks
|
| Biomaterial provider |
Central Drug Research Institute, India
|
| Treatment protocol |
Mice were treated with a single dose of hepatotoxicant
|
| Growth protocol |
Mice were kept under optimal conditions of light and temperature
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRI reagent (sigma)
|
| Label |
Cy5
|
| Label protocol |
CyScribe first strand cDNA labelling kit protocol (Amersham)
|
| |
|
| |
| Hybridization protocol |
The Cy5 and Cy3 labelled cDNAs were resuspended in Cyscribe Hyb buffer(Amersham) containing 10ug/ml sheared Salmon sperm DNA, and 10ug/ml Yeast tRNA.The samples were heated at 95 degree C for 3 minutes and immediately chilled to avoid secondary structures. The labelled samples were hybridized to the arrays and incubated for 16-18hrs at 42 degree C.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 with Molecular dynamics III scanner supported with ImageQuant v5.0.
|
| Description |
Mice (Mus musculus) were sacrificed and livers were snap frozen in liquid nitrogen, and subsequently stored at -80 degree C till further use. Liver samples were mechanically homogenized and total RNA was extracted with TRI reagent (Sigma). 25 ug of total RNA was converted into
labelled cDNA using CyScribe first strand cDNA labelling kit(Amersham). Briefly, a reaction mixture of 25uL contained 1uL oligo(dT) primers, 1uL dCTP mix, 1uL labelled CTPs, 2Ul; 0.1M DTT, 4UL; 5X RT buffer and 1uL CyScribe reverse transcriptase. Primer annealing was done at 70 degree C for 10 minutes followed by incubation at room temperature for
15 minutes. Primer extention was carried out at 42 degree C for 2 hours. Template RNA was degraded by 2uL; 2.5M NaOH at 37 degree C for 15 minutes. Reaction mixture was neutralized by adding 10uL; 2M HEPES buffer before purifying it from unincorporated nucleotides through GFX columns(Amersham). Purified labelled cDNA was concentrated by evaporation under vacuum after estimating the percent incorporation of the dyes spectrophotometrically.
|
| Data processing |
Image intensity data were extracted and analyzed with ArrayVision v8.0 analysis software. Background correted data was LOWESS normalized and log ratios were calculated using Avadis v4.3. Further, the data was processed to calculate mean log ratios for duplicate spots and analysed statistically either using Avadis v4.3 or Microsoft Excel to find differentially expressed genes. Multiple experiments were analysed with either Avadis v4.3 or TMEV v3.1(TIGR).
|
| |
|
| Submission date |
Mar 07, 2006 |
| Last update date |
May 24, 2006 |
| Contact name |
Sanjeev Noel |
| E-mail(s) |
sanjeevnoel@gmail.com
|
| Phone |
91-9935392997
|
| Organization name |
Central Drug Research Institute
|
| Department |
Toxicology
|
| Lab |
Genotoxicity
|
| Street address |
M G Marg
|
| City |
Lucknow |
| State/province |
Uttar Pradesh |
| ZIP/Postal code |
226001 |
| Country |
India |
| |
|
| Platform ID |
GPL3493 |
| Series (1) |
| GSE4874 |
Carbon tetrachloride and Acetaminophen hepatotoxicity study |
|
