|
| Status |
Public on Aug 06, 2010 |
| Title |
1st 2 hrs polysomes |
| Sample type |
other |
| |
|
| Channel 1 |
| Source name |
Polysomal RNA extracted from cardiac tissue treated with 10ml/kg saline
|
| Organism |
Mus musculus |
| Characteristics |
gender: Male
strain: C576B/AJ
age: 8 weeks
tissue: Heart
|
| Treatment protocol |
Mice randomly assigned to control or treatment group and treated with 10ml/kg saline (control) or 15mg/kg doxorubicin in 10ml/kg(treatment) as sacrificed at 0.5, 1, 2, 12, 24, 120 hours following dosing, hearts quickly removed and snap frozen in liquid nitrogen
|
| Growth protocol |
Mice housed with free assess to food and water, with a 12 hour light dark cycle
|
| Extracted molecule |
other |
| Extraction protocol |
Cardiac tissue lysated and subjected to centrifugation on a 0-65% sucrose gradient allowing separation of RNA species, gradients fractionated into 1ml fractions directly into Trizol, RNA extracted according to the manufactures instructions. Equal volumes of polysome or monosome fractions combined. Control monosome and treated monosome were assayed on one microarray slide and control polysome and treated polysomes assayed on another microarray
|
| Label |
Alexa 555
|
| Label protocol |
Upto 10µg monosome or polysome RNA were primed with 1µl of 8µg/µl oligo dT25N2 at 70 oC for 10 minutes, then reverse transcribed at 50oC for 2 hours in the presence of 1µl Superscript III (Invitrogen) and 100µM each dATP, dTTP, dGTP with 25 µM dCTP and 25µM cy-3 or cy-5
|
| |
|
| Channel 2 |
| Source name |
Polysomal RNA extracted from cardiac tissue treated with 15mg/kg doxorubcin in 10ml/kg saline
|
| Organism |
Mus musculus |
| Characteristics |
gender: Male
strain: C576B/AJ
age: 8 weeks
tissue: Heart
|
| Treatment protocol |
Mice randomly assigned to control or treatment group and treated with 10ml/kg saline (control) or 15mg/kg doxorubicin in 10ml/kg(treatment) as sacrificed at 0.5, 1, 2, 12, 24, 120 hours following dosing, hearts quickly removed and snap frozen in liquid nitrogen
|
| Growth protocol |
Mice housed with free assess to food and water, with a 12 hour light dark cycle
|
| Extracted molecule |
other |
| Extraction protocol |
Cardiac tissue lysated and subjected to centrifugation on a 0-65% sucrose gradient allowing separation of RNA species, gradients fractionated into 1ml fractions directly into Trizol, RNA extracted according to the manufactures instructions. Equal volumes of polysome or monosome fractions combined. Control monosome and treated monosome were assayed on one microarray slide and control polysome and treated polysomes assayed on another microarray
|
| Label |
Alexa 647
|
| Label protocol |
Upto 10µg monosome or polysome RNA were primed with 1µl of 8µg/µl oligo dT25N2 at 70 oC for 10 minutes, then reverse transcribed at 50oC for 2 hours in the presence of 1µl Superscript III (Invitrogen) and 100µM each dATP, dTTP, dGTP with 25 µM dCTP and 25µM cy-3 or cy-5
|
| |
|
| |
| Hybridization protocol |
4µg tRNA and 2x enhanced hybridization buffer (Genesiphere) were added, samples heated to 95oC for 2 minutes then 42oC for 30 minutes. Samples applied to microarrays enclosed in a Genetix humidified chamber. After hybridization overnight, slides were washed sequential.
|
| Scan protocol |
Scanned on a Axon 4200A scanner
Images were quantified using Genepix 6.0 software
|
| Description |
Samples normalized and A and B condensed into 1 file
|
| Data processing |
LOWESS normalized, log2 of processed red signal / green signal, A and B files combined, the Log2 for the monosome arrays for each gene was subtracted from that of the polysome array, the resultant Log2 represents differential translation. These data are present on the Series record.
|
| |
|
| Submission date |
Aug 26, 2009 |
| Last update date |
May 27, 2010 |
| Contact name |
Amy Pointon |
| Organization name |
MRC Toxicology Unit
|
| Department |
Systems Toxicology Group
|
| Street address |
Lancaster Road
|
| City |
Leicester |
| ZIP/Postal code |
LE1 9NH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL9106 |
| Series (2) |
| GSE17823 |
Mouse cardiac tissue, polysomes and monosomes: Vehicle treated control vs. 15mg/kg doxorubicin |
| GSE18459 |
Mouse cardiac tissues treated with doxorubicin and DMNQ |
|
