Real-time quantitative PCR analysis of human EBV-B cells during UPR. Real-time quantitative PCR analysis of human EBV-B cells during UPR

Human B cells were isolated from peripheral blood from one healthy donor and two patients with Common Variable Immunodeficiency (CVID) and were immortalized by infection with Epstein Barr virus (EBV) in vitro. Immortalized EBV-B cells (in biological triplicates) were treated twelve different ways for 4 hours: 1.untreated control, 2. Thapsigargin (Tg) 1 µM, 3. Tunicamycin (Tm) 10 µg/ml, 4. 4-phenylbutyrate (4-PBA) 1 mM, 5. Tg 1 µM plus 4-PBA 1 mM, 6. Tm 10 µg/ml plus 4-PBA 1 mM, 7. Dimethyl sulfoxide (DMSO) 1 mM, 8. Tg 1 µM plus DMSO 1 mM, 9. Tm 10 µg/ml plus DMSO 1 mM, 10. Tauroursodeoxycholic acid (TUDCA) 5 mM, 11. Tg 1 µM plus TUDCA 5 mM, and 12. Tm 10 µg/ml plus TUDCA 5 mM. We used Applied Biosystems TaqMan OpenArray Real-Time PCR System (Thermo Scientific) to quantitate gene expression of relevant genes from the cells (in technical triplicates). Overall design: qPCR gene expression profiling. EBV-immortalized B cells from three donors were used and treated separately as indicated in the summary. Equal amount total RNA from each donor was used for cDNA synthesis, and normalized amounts of cDNA were used for gene expression analysis (in technical triplicates). Samples were normalized to Gapdh expression. Fold changes were calculated as a ratio of T/NT (treated / non treated) expressed as log base 2.