|
| Status |
Public on Oct 14, 2015 |
| Title |
Liver_8mkd-furan_3wk-FFPE_sample42 [2-color array] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver_8mkd-furan_3wk-FFPE
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6C3F1
gender: female
exposed to: 8mg/kg bw furan for 3 wks
tissue: liver
sample type: FFPE (3wks in formalin)
|
| Treatment protocol |
Mice were dosed via oral gavage for 21 days. The furan dose was 8 mg/kg bw in cornoil. Control animals were gavaged with corn oil only. Tissues were collected 4 hours following the final dose.
|
| Growth protocol |
5-6 week old female specific pathogen free B6C3F1 mice were purchased from Charles River Breeding Laboratories (Portage, ME) and were allowed to acclimatize for at least 7 days prior to the start of the study. Feed (NIH-07; Zeigler Brothers, Inc., Gardners, PA) and tap water were available ad libitum. Mice were housed five per cage in polycarbonate cages in a specific pathogen free (SPF) and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility. All procedures were conducted in compliance with the Animal Welfare Act Regulations (9CFR1–4). Mice were handled and treated according to the guidelines provided in the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (ILAR, 1996; http://dels.nas.edu/ilar/).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were anaesthetized under carbon dioxide inhalation followed by exsanguination. Livers were removed and preserved in formalin for 18 hours or 3 weeks prior to being embedded in paraffin. Tissues were stored in paraffin blocks at room temperature until RNA extraction was performed. Total RNA was extracted from 1x10um section (18 hours in formalin) or 2x10um sections (3 weeks in formalin) using the FFPE RNeasy Midi Kit (Qiagen) 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 1.74-2.12 and RINs 1.3-2.4.
|
| Label |
Cy5
|
| Label protocol |
825 ng sample cDNA was labeled with 1.44 ml Cy5 dye and 825 ng of reference cDNA (made from a pool of all sample cDNA) was labeled with 1.24 μl Cy3 using the Genomic DNA ULS Labeling Kit (Agilent Technologies). Excess dye was removed using KREApure columns (Agilent Technologies), labeled product was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.), and degree of labeling was calculated using: Degree of labeling = [(340 x pmol per μl dye)/(ng per μl cDNA x 1000)] x 100% (with an acceptable range of 1.5-3%).
|
| |
|
| Channel 2 |
| Source name |
Pooled reference (all samples pooled together)
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6C3F1
gender: female
sample type: pooled reference
|
| Treatment protocol |
Mice were dosed via oral gavage for 21 days. The furan dose was 8 mg/kg bw in cornoil. Control animals were gavaged with corn oil only. Tissues were collected 4 hours following the final dose.
|
| Growth protocol |
5-6 week old female specific pathogen free B6C3F1 mice were purchased from Charles River Breeding Laboratories (Portage, ME) and were allowed to acclimatize for at least 7 days prior to the start of the study. Feed (NIH-07; Zeigler Brothers, Inc., Gardners, PA) and tap water were available ad libitum. Mice were housed five per cage in polycarbonate cages in a specific pathogen free (SPF) and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility. All procedures were conducted in compliance with the Animal Welfare Act Regulations (9CFR1–4). Mice were handled and treated according to the guidelines provided in the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals (ILAR, 1996; http://dels.nas.edu/ilar/).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were anaesthetized under carbon dioxide inhalation followed by exsanguination. Livers were removed and preserved in formalin for 18 hours or 3 weeks prior to being embedded in paraffin. Tissues were stored in paraffin blocks at room temperature until RNA extraction was performed. Total RNA was extracted from 1x10um section (18 hours in formalin) or 2x10um sections (3 weeks in formalin) using the FFPE RNeasy Midi Kit (Qiagen) 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 1.74-2.12 and RINs 1.3-2.4.
|
| Label |
Cy3
|
| Label protocol |
825 ng sample cDNA was labeled with 1.44 ml Cy5 dye and 825 ng of reference cDNA (made from a pool of all sample cDNA) was labeled with 1.24 μl Cy3 using the Genomic DNA ULS Labeling Kit (Agilent Technologies). Excess dye was removed using KREApure columns (Agilent Technologies), labeled product was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc.), and degree of labeling was calculated using: Degree of labeling = [(340 x pmol per μl dye)/(ng per μl cDNA x 1000)] x 100% (with an acceptable range of 1.5-3%).
|
| |
|
| |
| Hybridization protocol |
Hybridization mixtures were prepared using the Gene Expression Hybridization kit (Agilent Technologies) and CGHBlock (Agilent Technologies). 300 ng each of sample and reference cDNA was hybridized to SurePrint G3 Mouse GE 8x60K microarrays (Agilent Technologies). Hybridization occurred in a randomized block design, at 65°C for 17 hours at 20 rpm in the dark in an Agilent SureHyb hybridization chamber.
|
| Scan protocol |
Slides were washed with Wash Buffers 1 and 2 (Agilent Technologies) and scanned at 5 μm resolution on an Agilent G2505B scanner. Feature extraction was accomplished using Agilent feature extraction software version 11.
|
| Description |
3 weeks in formalin, FFPE samples
Sample 14
|
| 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 |
Oct 30, 2014 |
| Last update date |
Oct 15, 2015 |
| Contact name |
Anna Francina Jackson |
| E-mail(s) |
francina.jackson@hc-sc.gc.ca
|
| Organization name |
Health Canada
|
| Department |
Environmental Health Center
|
| Lab |
Dr. Carole Yauk
|
| Street address |
50 Colombine Drive, Tunney's Pasture
|
| City |
Ottawa |
| State/province |
Ontario |
| ZIP/Postal code |
K1A 0K9 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10787 |
| Series (2) |
| GSE62838 |
Global gene expression analysis of formalin-fixed paraffin-embedded (FFPE), furan-exposed mouse liver using two-colour Agilent microarrays |
| GSE62843 |
Global gene expression analysis in paired fresh-frozen and FFPE furan-exposed mouse liver samples |
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