Taqman glyceraldehyde 3 phosphate dehydrogenase gapdh

The mRNA expression levels of inflammatory cytokines were measured with the same methods employed in our previous studies using RT-PCR^{14 (link),19 (link)}. Total RNA was isolated from small intestinal tissue using an ISOGEN kit (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s protocol. Complementary DNA was produced using the High Capacity RNA-to-cDNA Kit (Life Technologies Corporation, Carlsbad, CA) according to the manufacturer’s protocol. Real-time quantitative RT-PCR analyses were performed using an Applied Biosystems 7500 Fast Real-Time PCR system and software (Life Technologies Corporation); the reaction mixture was prepared according to the manufacturer’s protocol using the TaqMan Fast Universal PCR master mixture (Life Technologies Corporation). The PCR thermal cycling conditions were: 45 cycles at 95 °C for 15 s and 60 °C for 1 min. The expression levels of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the small intestine and colon tissue were quantified using real-time RT-PCR, and standardized to TaqMan glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Life Technologies Corporation) mRNA levels. The expression levels of these mRNAs are indicated as ratios to the mean value of the vehicle-treated intestinal tissue. The primers and probes used for RT-PCR are detailed in Supplemental Table 1.

The local mRNA expression in the ileum was measured. Total RNA was isolated from the ileal tissue using the ISOGEN kit (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. Complementary DNA was produced using the high-capacity RNA-to-cDNA Kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. qRT-PCR analyses were performed using an Applied Biosystems 7500 Fast Real-Time PCR System and Software (Life Technologies). The reaction mixture was prepared according to the manufacturer’s protocol using the TaqMan Fast Universal PCR Master Mix (Life Technologies). The thermal cycling conditions were as follows: 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The expression levels of CRH, eotaxin-1, tpsb2, IL-5, IL-13, IL-4, and zonulin were quantified and standardized using the TaqMan glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Life Technologies) mRNA levels. The expression levels of these mRNAs are indicated as ratios relative to the mean value for the SS + PBS group. Primers and probes for CRH (Mm01293920_s1, Foster City, CA, USA), eotaxin-1 (Mm00441238_m1), tpsb2 (Mm01301240_g1), IL-5 (Mm00439646_m1), IL-13 (Mm00434204_m1), IL-4 (Mm00445259_m1), and zonulin (Mm00516884_m1) were purchased from Applied Biosystems.

Total RNA was prepared from the jejunum and ascending colon using an ISOGEN II kit (Nippon Gene Co., Ltd., Tokyo, Japan) following the manufacturer’s protocol. RNA was reverse transcribed into complementary DNA using a High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). qRT-PCR analyses were performed using an Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.). The abundance of mRNA of each gene was standardized to the levels of TaqMan glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific Inc.). The primers and probes used for qRT-PCR are listed in Table S1. Additionally, the protein expression level of zonula occludens (ZO)-1 in the jejunum and ascending colon was quantified by western blotting. The expression level of ZO-1 (61–7300, Invitrogen, Carlsbad, CA, USA) was normalized to that of β-actin. The detailed procedure has previously been described [26 (link)].