ELISA and Real-Time PCR
Collected blood samples were allowed to clot and centrifuged at 10,000
rpm for 10 min at 4°C to separate out serum. Standard sandwich ELISA was
performed to assess IgE levels (BD biosciences). For real-time PCR, RNA
was isolated from snap-frozen proximal colon lysates using TRIzol
extraction according to the manufacturers’ instruction. Real-time PCR
was performed on cDNA generated by using a Superscript II reverse
transcriptase kit (Life Technologies) with SYBR Green master mix
(Applied Biosystems) and commercially available primer sets (QIAGEN
QuantiTect). Samples were run on the ViiA7 real time PCR systems
(Applied Biosystems -Life Technologies).