gender: Female age: 6 months animal provider: Stratagene animal name: Big Blue Transgenic mice tissue: Liver agent: ENU time (days post-treatment): 7 days
Treatment protocol
The animal treatment protocol was described previously (Wang et al. 2004). Simply, six-month-old female mice were injected intraperitoneally with a single dose of 120 mg / kg body weight ENU (CAS# 759–73–9, Sigma, St. Louis, MO) or the vehicle dimethylsulfoxide (DMSO, Sigma). Groups of 4 or 5 animals were sacrificed on post-treatment days 1, 3, 7, 15, 30, and 120 for the ENU treatment and post-treatment days 1 and 30 for the control treatment (Figure 1). The tissues were isolated and frozen in -80C. The liver samples were used for this study. All animal experiments were conducted by following the recommendations set forth by our Institutional Animal Care and Use Committee.
Growth protocol
n/a
Extracted molecule
other
Extraction protocol
About 60 mg tissue was cut from each frozen liver sample and suspended in RNAlater-ICE (Ambion Inc., Austin, TX). The tissue pieces were transferred to 600 μl RNA lysis/binding buffer and minced using Tissue Tearor™ (Biospec products Inc., Bartlesville, OK). MiRNAs were isolated using mirVana™ miRNA isolation kit (Ambion) that was able to specifically enrich small RNAs with length of less than 200 nucleotides. The isolated RNAs were resolved in 100 μl nuclease-free water (Ambion). RNA concentrations were determined using Nanodrop 1000 Spectrophotometer (NanoDrop Technologies, Wilmington, Delaware). The quality of RNA samples were characterized on an Agilent BioAnalyzer (Agilent Technologies, Santa Clara, CA) using an RNA6000 Nano LabChip (Agilent).
Label
SYBR Green
Label protocol
Two hundred nanograms of enriched small RNA were converted into 1st strand cDNA using RT2 miRNA First Strand Kit (SABiosciences Corporation, Frederick, MD). The cDNAs were mixed with 2 × RT2 SYBR Green PCR Master Mix (SABiosciences).
Hybridization protocol
n/a
Scan protocol
n/a
Description
This mouse was sacrificed 7 days after ENU treatment small RNA with length less than 200 nucleotides PCR Array analysis of miRNA expression: Two hundred nanograms of enriched small RNA were converted into 1st strand cDNA using RT2 miRNA First Strand Kit (SABiosciences Corporation, Frederick, MD). The cDNAs were mixed with 2 × RT2 SYBR Green PCR Master Mix (SABiosciences) and dispersed into 384-well Mouse Genome miRNA PCR Array (MAM-3100A, SABiosciences) with 10 μl/well of the mixture. The PCR Array contained a panel of primer sets for 376 mouse miRNAs, four small RNAs as the internal controls and four qualification controls. The real-time qRT-PCR was performed on 7900 real-time PCR systems (Applied Biosystems Inc., Foster, CA) with the following cycling parameters: 95 0C for 10 mins, then 40 cycles of 950C for 15 s, 60 0C for 15 s and 72 0C for 30 s. SYBR Green fluorescence was recorded from every well during the annealing step of each cycle. The threshold cycle (Ct) value of each sample was calculated with software SDS 2.3 (Applied Biosystems). To calculate Cts, we set threshold line as 0.15 and kept it the same across all of the analyses. The baseline was automatically defined by the software.
Data processing
Normalization and statistical analysis were done in SABiosciences online PCR array data analysis web portal. Four genes, snoRNA251, snoRNA202, snoRNA142, and U6, were used as endogenous controls to calculate ∆Ct. Ttest’s were done between the ∆Ct’s of control group and the treatment group at each time point. The fold change (FC) for each miRNA was calculated using ∆∆Ct method. The formula: FC = 2^ [-(mean of ∆Ct values of treated samples - mean of ∆Ct values of control samples)] was used for up-regulated gene while FC= - 2^ (mean of ∆Ct values of treated samples - mean of ∆Ct values of control samples) was used for the down-regulations. miRNAs with p value < 0.01 and the absolute of FC > 2.0 were considered as differentially expressed miRNAs.
