Quantitative RT-PCR Analysis of Gene Expression

Total RNA was extracted from 2.0 to 5.0 × 10⁶ pelleted B cells using the RNeasy Mini Kit (Qiagen). Residual DNA was removed from the extracted RNA with gDNA eliminator columns (Qiagen). cDNA was synthesized from 1.0 to 2.0 μg of total RNA with the SuperScript III First-Strand Synthesis System (Invitrogen) using oligo-dT primer. Specific transcripts were measured by real-time qRT-PCR with appropriate primers as described (6 (link)). An Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) was used to measure SYBR Green (Bio-Rad Laboratories) incorporation with the following protocol: 95°C for 15 s, 40 cycles of 94°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Data acquisition was performed during a 72°C extension step. Melting curve analysis was performed from 72° to 95°C. The change in cycling threshold (ΔΔC_t) method was used to analyze levels of transcripts. Data were normalized to the expression level of β-ACTIN/β-Actin except noted otherwise (e.g., normalized to the Gapdh expression level).