Treatment of cell lines
U343 and HOG cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM, 61965-026, GIBCO™) supplemented with 10% fetal bovine serum (FBS, 10270-106, GIBCO™) and 1% penicillin-streptomycin solution (PEST, 15140-122, GIBCO™). Although some of the characteristics typical for oligodendrocytes and astrocytes may be changed in the glioma cells they are used for these experiments since it is not possible today to derive these cell types from a living human brain to perform this experiment. The cells were cultivated to about 30% confluence before treatment with antipsychotic drugs. Medium to treat the cells contained 1 μM Aripiprazole (A771000, Toronto Research Chemicals Inc.), 2 μM Clozapine (C6305, Sigma-Aldrich), 0.2 μM Haloperidol (H1512, Sigma-Aldrich), 0.25 μM Olanzapine (O253750, Toronto Research Chemicals Inc.) or 0.15 μM Risperidone (R525000, Toronto Research Chemicals Inc.), respectively, in DMEM supplemented with 10% FBS and 1% PEST. The concentrations used were in the typical range of blood plasma levels observed in patients treated with the drugs [9–12]. Three biological replicates were done for each test. The cells were treated for 6 or 24 hours. Control cells that were not treated, were incubated with the same medium without the antipsychotic agents, for 6 or 24 h. The cells were harvested by adding Trizol (Life Technologies, Sweden) directly to the cell plate after removing the medium, and the samples were collected in eppendorf tubes prior to RNA isolation.
In the initial experiment, we detected an increase of QKI-7 mRNA expression in Haloperidol treated U343 cells. To test whether the effect was dose dependent we performed an additional treatment with this drug using 10 times higher concentration (2 μM).
RNA isolation and Gene expression analysis
RNA was isolated according to standard Trizol extraction procedures [15]. The concentration of RNA was measured with a NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies). RNA samples were stored at -70°C prior to use.
Reverse transcription reactions included 1 μL RT-buffer (10×), 2.2 μL MgCl2 (25 mM), 2 μL dNTPs (10 mM), 0.5 μL oligo dT (10 μg/L), 0.2 μL RNase inhibitor, 0.25 μL reverse transcriptase (Taqman Reverse Transcription Reagents, N808-0234, Applied Biosystem) and 3.85 μL of sample (about 500 ng of RNA). These reactions were incubated at 25°C for 10 min and 48°C for 1 hour.
Real-time RT-PCR was performed with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, USA) as follows: 2 minutes at 50°C and 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Each reaction was carried out in a total volume of 25 μL, consisting of 9.8 μL Power SYBR® PCR Master mix (Applied Biosystems, Foster City, USA), 0.3 μM of each primer (Thermo Electron Cooperation, Germany) and ~10–100 ng of cDNA. The primer for the reference gene (actin-β, ACTB) as well as for the QKI splice variants, QKI-5, QKI-6, QKI-7, were uniquely designed using Primer Express (Applied Biosystems, Foster City, USA), as described previously [2] (see Additional file 2 for primer sequences). The expression data was collected and analyzed with the ABI PRISM 7000 SDS software (Applied Biosystems, Foster City, USA).
Statistical analysis
Messenger RNA expression levels of the QKI splice variants were normalized with the expression levels of the endogenous control ACTB. In other words, our target variable for the statistical analysis was the difference between QKI and ACTB expression on a logarithmic scale. To test the effect of antipsychotic treatment on QKI mRNA expression, we analyzed each cell line separately with a two-way ANOVA model, which included the factors treatment, time and the interaction between treatment and time. Comparisons between treated cells and their corresponding controls were carried out with linear contrasts, (i.e. with pre-planned t-test utilizing the pooled estimate of error variance). The statistical analysis was carried out in Proc GLM (SAS/STAT software, version 9.1.3, SAS institute Inc., Cary, NC).