Gene Expression Analysis in Lymphoid Tissues

The total RNA was isolated from frozen jejunum, spleen, thymus, and bursa, and then the concentration was determined using Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA). Following purification, RNA (approximately 200 ng) was used as template for reverse transcriptase reactions using a reverse transcript kit (Takara, Japan). The resulting cDNA was immediately profiled for mRNA expression using a 7900 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were selected as the reference genes, and relative gene expression was calculated as previously described [31 (link)]. Primer sequences for all genes are provided in Table S2.