|
| Status |
Public on Sep 27, 2013 |
| Title |
75 mg/kg Sample 17 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
75 mg/kg
|
| Organism |
Mus musculus |
| Characteristics |
strain: MutaTMMouse
tissue: Forestomach
gender: Male
|
| Treatment protocol |
Mice were dosed via oral gavage daily for 28 consecutive days. Three different doses of BaP dissolved in olive oil were selected to dose the animals: 25 mg/kg BW, 50 mg/kg BW, and 75 mg/kg BW. Control animals were gavaged with olive oil only. Tissues were collected 72 hours following the final dose.
|
| Growth protocol |
Male MutaTMMouse, aged 24-26 weeks, were individually housed in a plastic film isolator, provided with water and food (2012 Teklad Global standard rodent diet) ad libitum, and were subjected to a 12 hour light/dark cycle. All animal procedures were in accordance with the guidelines of the Canadian Council for Animal Care and approved by the Health Canada Animal Care Committee.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were anaesthetized under isofluorane followed by exsanguination. Murine forestomachs were removed, placed in cryovials and immediately snap frozen in liquid nitrogen. Tissues were stored at -80oC until RNA extraction was performed. Total RNA was extracted from random sections (n=5 per group) using Trizol reagent (Invitrogen) and further purified using the RNeasy Mini Kit (Qiagen) with an on-column DNase I digestion according to the manufacturer's instructions. Total RNA concentration and quality was determined using a Nanodrop 1000 Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. RNA samples used for gene expression microarray had A260/280 ratios > 2.0 and RINs > 6.6.
|
| Label |
Cy5
|
| Label protocol |
Total RNA (200ng per array) was labeled using the Agilent Quick Amp Labeling Kit, Two-Color (Agilent Cat #: 5190-0444), according to the manufacturer's instructions. Control and experimental samples were labeled with Cyanine 5-CTP; whereas universal mouse reference RNA was labeled with Cyanine 3-CTP.
|
| |
|
| Channel 2 |
| Source name |
Universal Mouse Reference RNA
|
| Organism |
Mus musculus |
| Characteristics |
tissue: 11 mouse cell lines
|
| Treatment protocol |
Mice were dosed via oral gavage daily for 28 consecutive days. Three different doses of BaP dissolved in olive oil were selected to dose the animals: 25 mg/kg BW, 50 mg/kg BW, and 75 mg/kg BW. Control animals were gavaged with olive oil only. Tissues were collected 72 hours following the final dose.
|
| Growth protocol |
Male MutaTMMouse, aged 24-26 weeks, were individually housed in a plastic film isolator, provided with water and food (2012 Teklad Global standard rodent diet) ad libitum, and were subjected to a 12 hour light/dark cycle. All animal procedures were in accordance with the guidelines of the Canadian Council for Animal Care and approved by the Health Canada Animal Care Committee.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were anaesthetized under isofluorane followed by exsanguination. Murine forestomachs were removed, placed in cryovials and immediately snap frozen in liquid nitrogen. Tissues were stored at -80oC until RNA extraction was performed. Total RNA was extracted from random sections (n=5 per group) using Trizol reagent (Invitrogen) and further purified using the RNeasy Mini Kit (Qiagen) with an on-column DNase I digestion according to the manufacturer's instructions. Total RNA concentration and quality was determined using a Nanodrop 1000 Spectrophotometer and an Agilent 2100 Bioanalyzer, respectively. RNA samples used for gene expression microarray had A260/280 ratios > 2.0 and RINs > 6.6.
|
| Label |
Cy3
|
| Label protocol |
Total RNA (200ng per array) was labeled using the Agilent Quick Amp Labeling Kit, Two-Color (Agilent Cat #: 5190-0444), according to the manufacturer's instructions. Control and experimental samples were labeled with Cyanine 5-CTP; whereas universal mouse reference RNA was labeled with Cyanine 3-CTP.
|
| |
|
| |
| Hybridization protocol |
Equal amounts of Cy5-labeled experimental cRNA and Cy3-labeled reference RNA (300ng) were co-hybridized to Agilent Sureprint G3 Mouse GE 8x60K microarray slides, according to the instructions outlined in the Agilent Two-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) Protocol. Samples were hybridized for 17 hours at 65oC and 10 rpm.
|
| Scan protocol |
Microarray slides were scanned using an Agilent DNA Microarray Scanner (G2505B) according to manufacturer's recommendations. Data was extracted from the scanned images using Feature Extraction Version 10.7.3.1.
|
| Data processing |
The log2 of the ratio (sample(Cy5)/reference(Cy3)), using the median signal intensities were normalized using lowess using the transform.madata function in the maanova library in R.
|
| |
|
| Submission date |
Jan 11, 2013 |
| Last update date |
Sep 27, 2013 |
| Contact name |
Sarah Labib |
| Organization name |
Health Canada
|
| Street address |
204b-50 Colombine
|
| City |
Ottawa |
| State/province |
Ontario |
| ZIP/Postal code |
K1A0K9 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE43438 |
Sub-chronic forestomach transcriptional response in adult male MutaTMMouse following oral exposure to benzo(a)pyrene |
|
