Quantitative RT-PCR Analysis of EBI3 and p35 Expression

qRT-PCR analysis was carried out on an ABI step one plus real-time system (Applied Biosystems). We used a Qiagen RNeasy Kit (Qiagen, Valencia, CA) to isolate total cellular RNA from each sample. One microgram of total RNA from each sample was subjected to cDNA synthesis using the Superscript III reverse transcriptase (Invitrogen). cDNAs were amplified by PCR with primers as following: EBI3 (sense, 5′-CATTGCCACTTACAGGCTCG-3′; antisense, 5′-GGATGT ACGATTTACAGTGACGT-3′); p35 (sense, 5′-CAATCACGCTACCTCCTCTTT T-3′; antisense, 5′-CTTTGTAATAAGGACGTGACGAC-3′); β-actin (sense, 5′-GGCTGT ATTCCCCTCCATCG-3′; antisense, 5′-TGTACCGTAACAATGGTTGACC-3′). The ratio of each interest gene to β-actin for each sample was noted as mRNA expressive level of the interest genes. SYBR green quantitative PCR amplifications were performed in the Step one plus Detection System (Applied Biosystems). The comparative Ct (ΔΔCt) method was adopted to definite the expression fold change [18 (link),19 (link)]. All the primers were synthesized by Gene and Technology of China in Shanghai.