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).