Mouse was injected one i.p. injection of 40 mg/kg PCN in corn oil in the morning. After 24 hours animal was euthanized using CO2 suffocation.
Growth protocol
Mouse was housed in normal light-cycle room, maintained on standard rodent chow (diet 3430, Provimi Kliba SA, Kaiseraugst, CH), and were allowed water and food ad libitum. One week prior treatment diet was switched to a 1% (w/w) cholesterol diet (diet Western 24769, Provimi Kliba SA, Kaiseraugst, CH).
Extracted molecule
total RNA
Extraction protocol
Left lateral lobes of livers were snap frozen in liquid nitrogen and stored at -80ºC. Total RNA was isolated using TRI-reagent (Sigma, St Louis, MI, USA) according to the manufacturer’s protocol and purified by ethanol precipitation.
Label
Cy3
Label protocol
To 20 μg of sample total RNA we added spike in RNA: 250 pg of Firefly Luciferase mRNA (Promega, Madison, WI, USA), and 0.5 μL of test spike mix from Lucidea Universal ScoreCard kit. We reverse-transcribed mRNA to amino-allyl cDNA using 2.5 μg of Oligo dT (Invitrogen, Carlsbad, CA, USA), 400U of SuperScriptTM III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and 1μL of 10mM amino-allyl dUTP (Sigma, St Louis, MI, USA) according to the manufacturer’s protocol. Reaction was stopped after 2 hours by adding 10 μL of 0.5 M EDTA and of 1M NaOH and incubation at 65ºC for 15 min. 10 μL of 1 M HCl was added and cDNA was purified using MinElute PCR Purification Kit (Qiagen GmBH, Hilden, D) according to manufacturer’s protocol with exception of using a phosphate buffer (5mM KPO4 pH 8.5 in 80% ethanol) for washing step and MilliQ water for elution. Purified amino-allyl cDNA was dried and resuspended in 4.5 μL of 0.2 M Na2CO3 (pH 9.0) and 4.5 μL of cyanin dye in DMSO (Amersham Biosciences, GE Healthcare UK limited, Little Chalfont, UK). Labeling reaction was incubated at room temperature for two hours. Next we added 35 μL of 0.1 M Na acetate (pH 5.2) and purified reaction using MinElute PCR Purification Kit (Qiagen GmBH, Hilden, D) according to manufacturer’s protocol. Labeled cDNA was eluted in water and 1 μL was used for analyses using ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, Delaware, USA).
Mix of all samples used in experiment: mice treated with vehicle, TCPOBOP and PCN.
Growth protocol
Mix of all samples used in experiment: mice on standard and cholesterol diet.
Extracted molecule
total RNA
Extraction protocol
Mix of all samples used in experiment: all isolated using TRI-reagent (Sigma, St Louis, MI, USA) according to the manufacturer’s protocol and purified by ethanol precipitation.
Label
Cy5
Label protocol
Labeling protocol is the same as for channel 1 with exception of adding a reference spike mix from Lucidea Universal ScoreCard kit.
Hybridization protocol
Channel 1 sample was mixed with channel 2 sample hybridization buffer was added (final concentration was 3xSSC, 0.2% SDS) and samples were denatured. Using LifterSlip cover glasses (Erie Scientific Company, Portsmouth, NH, USA) samples were hybridized for 16h at 65ºC in a water bath using humidified hybridization chambers (HybChambers, GeneMachines, San Carlos, CA, USA). Prior hybridizations Steroltalk v2 slides were pre-hybridized for 1 hour at 42ºC in 5xSSC, 0.1%SDS and 1%BSA, then washed at room temperature for 5 min in 2xSSC and 0.1%SDS, 3 min in 0.2xSSC and 2 min in MilliQ water and dried by centrifugation. After hybridization slides were washed at room temperature for 5 min in 2xSSC and 0.5%SDS, 5 min in 0.5xSSC, 5 min in 0.05xSSC, and then dried using vacuum centrifuge.
Scan protocol
Slides were scanned using Tecan LS200 scanner (Tecan Group Ltd., Maennedorf, CH). Images were analyzed using Array-Pro Analyzer 4.5 (Media Cybernetics, Bethesda, MD, USA). The median feature and local background intensities were extracted together with the estimates of their standard deviation
Description
No additional data.
Data processing
Data filtration and normalization was done in Orange (http://magix.fri.uni-lj.si/orange/). Only features with foreground to background ratio higher than 1.5 and coefficient of variation (CV, ratio between standard deviation of the background and the median feature intensity) lower than 0.5 in both channels were used for further analysis, other were filtered out. Log2 ratios were normalized using LOWESS fit to spike in control RNAs according to their average intensity. For normalization, two types of spike in controls were used: custom-made Firefly luciferase and commercial Lucidea Universal ScoreCard.
