Quantitative real-time RT-PCR analysis
Total RNA was extracted from CD27+IgD+ B cells using RNeasy Mini Kit (Qiagen, Hilden, Germany), and reverse transcribed into cDNA with the RevertAid First Strand cDNA synthesis kit (Fermentas, Glen Burnie, MD, USA) according to the manufacturer’s instructions. The resulting cDNA was subjected to real-time PCR analyses. Gene expression was quantified relative to the expression of the housekeeping gene GAPDH as well as β-actin, and was normalized to control by standard 2-ΔΔCT calculation(11).