Quantifying NF-κB2, RelB, NIK, and Bcl3 Gene Expression

Expression levels of the NF-κB2, RelB, NIK and Bcl3 genes were quantified by real-time PCR (qPCR) assays. Specific primers and probes for NF-κB2, RelB, NIK, Bcl3 and IPO8 genes (Importin 8 was used as a reference gene) were designed to bind to all isoforms using OligoAnalyzer 3.1 (Integrated DNA Technologies, Inc.)^{52 (link)}, according to the sequences provided in NCBI (http://www.ncbi.nlm.nih.gov/). Primers and probes were synthesized by IDT. Primer sequences and reaction conditions can be provided upon request. The qPCR reactions were carried out in triplicates, in a total volume of 20 μl, containing 5 μl of cDNA in 1× Kapa Probe Fast Master Mix (KAPA BIOSYSTEMS, Woburn, MA, USA) in an MX3000p cycler (Stratagene, La Jolla, CA, USA). Relative expression levels were calculated using the LinReg Program^{53 (link)} and were normalized to levels obtained for the calibrator sample and to IPO8 levels.