Todorčević et al. BMC Genomics 2010, 11:39 http://www.biomedcentral.com/1471-2164/11/39 background from median signal intensities, the expres- sion ratios were calculated. Lowess normalization was performed first for the whole slide and next for twelve rows and four columns per slide. The differential expression was assessed by difference of the mean log- expression ratios between the slides with reverse label- ling (6 spot replicates per gene on each slide, Student’s t-test, p < 0.01). Complete microarray results are sub- mitted to NCBI GEO Omnibus (GSE18389). Quantitative real-time RT-PCR RNA isolated at days 1, 4, 5, 6, 7, 9, 10, 11, 12, 15, 20, 23 and 30 was used for qPCR. Approximately 200 ng of total RNA was reverse-transcribed into cDNA using TaqMan® Gold RT-PCR Kit (Applied Biosystems, Foster City, CA, USA), a 25 μl reaction system according to the manufacturer’s protocol. The PCR primers (Table 1) were designed using the Vector NTI (Invitrogen, Carls- bad, CA, USA) and synthesized by Invitrogen. Efficiency was checked from tenfold serial dilutions of cDNA for each primer pair. A 2 × SYBR® Green PCR Mastermix (Roche Diagnostics, Mannheim, Germany), 0.4 μM of each primer, and the cDNA template were mixed in 12 μl volumes. PCR was performed in duplicates in 96-well optical plates on Light Cycler 480 (Roche Diagnostics, Mannheim, Germany). Different controls were used for microarray and qPCR. All time points were compared relative to day 1 in qPCR analyses. Relative expression of mRNA was calculated using the ΔΔCt method. Three commonly used genes (18S, elongation factor 1A and eukaryotic translation initiation factor 3, subunit 6) were tested for stability using the GeNorm and NormFinder. Finally, elongation factor 1A (EF1A) met criteria of sta- bility in the analyzed material. Differences between con- trol and cells at different developmental stages were assessed with Student’s t-test (p < 0.05). Cell proliferation Cell proliferation was assessed by the immunocytochem- ical detection of PCNA)(Zymed Laboratories, South San Francisco, CA, USA). Cells were washed in PBS and fixed in 70% ethanol for 30 minutes at 4°C. Endogenous peroxidise activity was blocked with with 3% hydrogen peroxide in methanol for 10 min. The cells were washed three times with in PBS, then incubated with a mouse anti PCNA monoclonal antibody (clone PC10) using a PCNA immuno detection kit, following the manufac- turer’s instructions. The cells were counterstained with Mayer’s haematoxylin for 2 min, washed in water, dehy- drated in a graded series of alcohol solutions, cleared with xylene, and mounted with Histomount. PCNA-con- taining nuclei were stained dark brown. Two hundred cells were observed and the percentage of proliferating cell was calculated. Page 15 of 17 Lipid accumulation in adipocytes The quantity of lipids in cytoplasm were estimated by oil-red O staining according to Ramirez-Zacarias et al. [70] on days 1, 4, 7, 9, 15 and 30. Briefly, cells were washed twice with PBS, fixed with 10% cold formalin for 30 min, rinsed in water and stained for 2 h with fil- tered oil red O in isopropanol at room temperature. For relative quantitative measurements of TAGs accumula- tion, bottles were washed with PBS to remove excess of stain solution, dried, the colour was dissolved in 100% isopropanol and the absorbance was measured spectro- photometrically in a Victor 3 microplate reader Perki- nElmer (Wellesley, MA, USA) at 500 nm. Superoxide dismutase assay SOD activity was assayed with a kit (Cayman Chemicals, Ann Arbor, MI, USA), which utilizes a tetrazolium salt to detect superoxide radicals generated by xanthine oxi- dase and hypoxanthine. Colour was measured at 405 nm in a Titertek Multiskan PLUS MKII (Labsystems, Helsinki, Finland) plate reader. One unit of SOD was defined as the amount of enzyme needed to achieve 50% dismutation of the superoxide radical. Acknowledgements We thank Inger Ø. Kristiansen for her skilful technical assistance and Turku Centre of Biotechnology for the preparation of microarrays. This study was carried out with support from Norwegian Research Council (NRC). Author details 1Nofima, Norwegian Institute of Food, Fisheries and Aquaculture Research, P. O. Box 5010, Ås NO-1430, Norway. 2Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, Ås NO-1432, Norway. Authors’ contributions MT and BR contributed to the overall experimental design. SS and AK carried out microarray analyses. MT carried out cell culture work, and assay analyses. MT and SS carried out qPCR analyses and produced the first manuscript draft. 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