Reverse Transcription PCR
Total RNA was extracted using lysis buffer (RNeasy kit; Qiagen, Crawley, UK). During RNA purification, genomic DNA was digested with RNase-free DNase (Amersham Biosciences). Next, 0.5 µg of total RNA was reversed transcribed using the avian myeloblastosis virus RT (Promega, Southampton, UK). For relative quantification, RT-PCR was carried out using cDNA probes. Primers for IL-4 were from Sigma-Genosys (Cambridge, UK). Sequences of GADPH used are as follows: forward 5′-CCACCCATGGCAAATTCCATGGC, reverse 3′-TCTAGACGGCAGGTCAGGTCCAC.