|
| Status |
Public on Oct 18, 2012 |
| Title |
Rat liver LE 10 TCDD 19 hours rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
liver, Long-Evans, 10 TCDD, 19 hour exposure
|
| Organism |
Rattus norvegicus |
| Characteristics |
gender: Male
tissue: Liver
strain: Long-Evans
age: 10-12 weeks
treatment: 10 ug/kg TCDD
exposure: 19 hours
|
| Treatment protocol |
Animals were treated with either TCDD or corn oil vehicle alone by oral gavage.
|
| Growth protocol |
Animals were housed in suspended, stainless-steel wire-mesh cages in groups of 4 rats with a 12h/12h light/dark cycle and with pelleted R36 feed and tap water. The temperature was maintained at 21 +/- 1 degree Celcius with relative humidity 50% +/- 10%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Qiagen RNeasy Mini kits according to the manufacturer’s instructions (Qiagen, Mississauga, Canada). RNA was quantified using a NanoDrop UV spectrophotometer (Thermo Scientific, Mississauga, ON) and RNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
FAM
|
| Label protocol |
RNA was reverse-transcribed using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Carlsbad, CA) following the manufacturer’s instructions, with the thermal cycler set to run at 25°C for 10 minutes, 37°C for 2 hours, 85°C for 5 minutes followed by a rapid cooling to 4°C. qPCR was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA) using the ΔΔCT standard 384-well PCR plate settings; an initial 2-minute hold at 50°C was performed to ensure that any UTP-containing contaminants from previous PCR reactions were fully digested by the AmpErase UNG nuclease. Polymerase was activated by a 10-minute hold at 95°C and reactions carried out by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C
|
| |
|
| Hybridization protocol |
n/a
|
| Scan protocol |
n/a
|
| Description |
Treated
|
| Data processing |
Raw qPCR data (Ct values) were loaded into the R statistical environment (v2.12.2). Wells flagged with Non-Amplified Well were removed prior to downstream analysis. Data was normalized using reference genes Gapdh and Pgk1 and fold-changes calculated using the mean of the biological replicates (Treated - Control, in log2 space). Matrix non-normalized contains the mean of the raw Ct values. Matrix normalized contains values normalized to the housekeeping genes (delta Ct). Fold Change contains values of treated animals compared to untreated animals (Ct[treated] - Ct[untreated]).
|
| |
|
| Submission date |
Oct 05, 2012 |
| Last update date |
Oct 18, 2012 |
| Contact name |
Stephenie Prokopec |
| E-mail(s) |
[email protected]
|
| Organization name |
Ontario Institute for Cancer Research
|
| Street address |
661 University Avenue, Suite 510
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL16146 |
| Series (2) |
| GSE41388 |
Systematic Evaluation of Medium-Throughput mRNA Abundance Platforms |
| GSE43251 |
NanoString analysis of a rat liver mRNA panel following TCDD treatment |
|
