Enterocyte Responses to LPS and BG

To explore the impact of LPS and BG on enterocyte responses, the RNA from stimulated Caco-2 cells was prepared using FavorPrep Tissue total RNA purification Mini Kit (Favorgen Biotech Corp, Vienna, Austria) and cDNA was synthesized by cDNA Synthesis assay (Thermo Fisher Scientific, Wilmington, DE, USA) before the detection by SYBR green-based real-time polymerase chain reaction (PCR) (Thermo Fisher Scientific, Wilmington, DE, USA). The oligonucleotide primers for the experiment were (i) Toll-like receptor 2 (TLR-2); forward 3′-TCCTCCAATCAGGCTTCTCTGTCTT-5′ and reverse 3′-CTCGCAGTTCCAAACATTCC-5′, (ii) Toll-like receptor 4 (TLR-4); forward 3′-CACAGACTTGCGGGTTCTAC-5′ and reverse 3′-AGGACCGACACACCAATGATG-5′, (iii) Dectin-1; forward 5′-GAACCACAGTCAACCCACAC-3′ and reverse 5′-CCAGTTGCCAGCATTGTCTT-3′, and (iv) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; a house-keeping gene); forward 5′-GTGAAGGTCGGTGTCAACGGATTT-3′ and reverse 5′-CACAGTCTTCTGAGTGGCAGTGAT-3′ following the previous protocol^{36 (link)}. The results were demonstrated in terms of relative quantitation of the comparative threshold (delta-delta Ct) method (2 − ∆∆Ct) as normalized by GAPDH.