Favorprep tissue total rna purification mini kit

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.

For gene expression, cells were collected at day 7 (MSCs) or 14 (HPAs) of differentiation and total RNA was extracted using the High Pure RNA Isolation kit (Roche Diagnostics, Switzerland). For tissues, mRNA was extracted using the FavorPrep™ Tissue Total RNA Purification Mini Kit (Favorgen Biotech Corp., Ping-Tung, Taiwan) and Qiagen RNeasy Lipid Kit (Qiagen, Hilden, Germany). For real-time PCR analysis, RNA was reverse transcribed and the HPRT or GAPDH gene was used to standardize mRNA expression in each sample. The primers used in this study are indicated in Supplementary Table S2.