After overnight culture, the medium was replaced by serum-free medium. Forty-eight hours after serum deprivation, cells were cultured in the presence or absence of inducers for 12, 24 or 48 hours. Hepatocytes were treated with rifampicin (5 µM), atorvastatin (10 µM) or rosuvastatin (10 µM).
Growth protocol
Human livers from donors 089, 114, 129, 269, 270, 271 and 272 were obtained from kidney transplant donors or from lobectomy segments resected from adult patients. Liver cells were isolated by the method of Bayliss and Skett (1996). Hepatocytes having viability better than 90% as determined by trypan blue exclusion, were used in the experiments. The cells were plated at a density of 1.7x105 cells/cm2 in plastic dishes precoated with collagen in medium described by Ferrini et al. (1998).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from human hepatocytes using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA was precipitated using ethanol.
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).
Channel 2
Source name
Mix of untreated cells from all donors and other human liver RNA samples [reference]
After overnight culture, the medium was replaced by serum-free medium. Forty-eight hours after serum deprivation, cells were cultured in the presence or absence of inducers for 12, 24 or 48 hours. Hepatocytes were treated with rifampicin (5 µM), atorvastatin (10 µM) or rosuvastatin (10 µM).
Growth protocol
Human livers from donors 089, 114, 129, 269, 270, 271 and 272 were obtained from kidney transplant donors or from lobectomy segments resected from adult patients. Liver cells were isolated by the method of Bayliss and Skett (1996). Hepatocytes having viability better than 90% as determined by trypan blue exclusion, were used in the experiments. The cells were plated at a density of 1.7x105 cells/cm2 in plastic dishes precoated with collagen in medium described by Ferrini et al. (1998).
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from human hepatocytes using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA was precipitated using ethanol.
Label
Cy5
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).
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
Arrays scanned on an Tecan LS200 scanner; images analyzed using MediaCybernetics Array-Pro Analyzer software.
Description
Human liver cells treated with atorvastatin for 48h, donor d129.
Data processing
Raw data deposited to BASE (BioArray Software Environment). Normalization is achieved through expression of selected spike-in controls using OWNormalize widget from Orange (a component based machine learning library for Python developed at Laboratory of Artificial Intelligence, Faculty of Computer and Information Science, University of Ljubljana, Slovenia). Since Steroltalk arrays are small-scale, containing about 300 genes which were selected from specific pathways, it is expected that their median expression varies across the experimental conditions.
