strain: B6C3F1 developmental stage: adult gender: male tissue: lung treatment group: Control
Treatment protocol
Age matched adult male B6C3F1 mice (27-30 d, Charles Rivers Laboratories, St Constant, Quebec, Canada) were housed individually under a 12:12 h light: dark cycle with food and water available ad libitum. Mice were randomly assigned (6 per group) to a control or treatment group. Mice were treated with a daily dose of BaP in corn oil with 5, 50, 150 or 300 mg/kg (oral gavage, 10 ml/kg) for three consecutive days. Control mice received corn oil only. Mice were anaesthetized under isofluorane and sacrificed by exsanguination 4 or 24 hours after the last treatment. Right and left lung lobes were removed and immediately snap frozen and stored at -80ºC until use. Blood serum was collected as described below. 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
The mirVana miRNA Isolation Kit (Ambion, Streetsville, ON, Canada) was used to isolate RNA from random left lung sections, according to the manufacturer’s protocol and as described in [29]. RNA quality and quantity was determined as described above.
Label
Cy3
Label protocol
Freshly isolated individual lung total RNA samples (100 ng, n=5/group) from control and treated groups (150 and 300 mg/kg, 4 hour group) were dephosphorylated by incubation with calf intestinal phosphatase at 37ºC for 30 min, denatured using 100 % DMSO at 100ºC for 5 min, then labelled with pCp-Cy3 using T4 ligase by incubation at 16ºC for 1 hour (Agilent miRNA Complete Labelling and Hybridisation Kit, Agilent Tech, Mississauga, ON, Canada). Labelled RNA samples were hybridised to an individual array on 8 x 15K format Agilent mouse miRNA array slides, with each array containing probes for 567 mouse miRNAs and 10 mouse gamma herpes virus miRNAs. Hybridisations were performed in SureHyb chambers (Agilent) for 20 hours at 55ºC and washed according to the manufacturer’s instructions. Arrays were scanned at a resolution of 5 m using an Agilent G2505B scanner and data were acquired using Agilent Feature Extraction software version 9.5.3.1.
Hybridization protocol
Freshly isolated individual lung total RNA samples (100 ng, n=5/group) from control and treated groups (150 and 300 mg/kg, 4 hour group) were dephosphorylated by incubation with calf intestinal phosphatase at 37ºC for 30 min, denatured using 100 % DMSO at 100ºC for 5 min, then labelled with pCp-Cy3 using T4 ligase by incubation at 16ºC for 1 hour (Agilent miRNA Complete Labelling and Hybridisation Kit, Agilent Tech, Mississauga, ON, Canada). Labelled RNA samples were hybridised to an individual array on 8 x 15K format Agilent mouse miRNA array slides, with each array containing probes for 567 mouse miRNAs and 10 mouse gamma herpes virus miRNAs. Hybridisations were performed in SureHyb chambers (Agilent) for 20 hours at 55ºC and washed according to the manufacturer’s instructions. Arrays were scanned at a resolution of 5 m using an Agilent G2505B scanner and data were acquired using Agilent Feature Extraction software version 9.5.3.1.
Scan protocol
Arrays were scanned at a resolution of 5 um using an Agilent G2505B scanner and data were acquired using Agilent Feature Extraction software version 9.5.3.1.
Description
microRNA
Data processing
Non-background subtracted raw data were quantile normalized [56]. Present calls were determined as signals that were greater than 3 trimmed SDs above the trimmed mean of the (-)3xSLv1 probes on the array. Technical replicates for individual miRNAs were averaged using the median signal intensity. Boxplots and cluster analyses were used to identify potential outliers (poor quality chips). This quality control check resulted in the elimination of one array from the analysis. Identification of differentially expressed miRNAs was carried out on the probe level as well as the miRNA level. The MAANOVA model included the sample identity as a random effect and the gene specific variance estimate (F1 Test) was used to test for differences between the controls and treated samples. In this analysis, parametric p-values were obtained and were then FDR corrected. Cy3 is the median of the technical replicates of the normalized signal intensity. Flag = 1 indicates if the median signal intensity was absent.
