Reverse transcription buffer

Total RNA was extracted using Sepasol RNAI Super (Nacalai Tesque, Inc.), as described by the manufacturer's instructions. The extracted total RNA samples were dissolved in TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) and stored at −80°C until use. The RNA samples were treated with RQ1 RNase-free DNase mixture (Promega Co., Madison, WI, USA; 1 µg total RNA, 1× DNase buffer, and 1 UDNase in 10 µl) on a programmable thermal controller (PTC-100; MJ Research, Waltham, MA, USA), programmed at 37°C for 30 min and then at 65°C for 10 min with 1URQ1 DNase Stop Solution (Promega Co.). The concentration of RNA in each sample was measured using Nano Drop Lite (Thermo Fisher Scientific., Waltham, MA, USA). The RNA samples were reverse-transcribed using Rever-Tra Ace (Toyobo Co., Ltd., Osaka, Japan) as per manufacturer's instructions. The reaction mixture (10 µl) comprised 0.5 µg total RNA, 1× reverse transcription buffer (Toyobo Co., Ltd.), 1 µM deoxyribonucleotide triphosphate (dNTP) mixture (Toyobo Co., Ltd.), 5 URNase inhibitor (Toyobo Co. Ltd.), 0.25 µg of oligo (dt) 20 (Toyobo Co., Ltd.), and 50U Rever Tra Ace. The reverse transcription was performed at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min using a programmable thermal controller. Finally, the cDNA samples were stored at −20°C until use.

Total RNA was extracted using Sepasol RNA Super (Nacalai Tesque, Inc., Kyoto, Japan), following the manufacturer's instructions. The extracted total RNA samples were dissolved in TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) and stored at −80°C until required. Samples were treated with RQ1 RNase-free DNase mixture (Promega Co., Madison, WI; 1-μg total RNA, 1 × DNase buffer, and 1 unit DNase in 10 μL) on a programmable thermal controller (PTC-100; MJ Research, Waltham, MA), programmed at 37°C for 30 min, followed by incubation at 65°C for 10 min with 1 U RQ1 DNase Stop Solution (Promega Co.). The concentration of RNA in each sample was measured using NanoDrop Lite (Thermo Fisher Scientific., Waltham, MA). The RNA samples were reverse-transcribed using ReverTra Ace (Toyobo Co., Ltd., Osaka, Japan) as per manufacturer's instructions. The reaction mixture (10 μL) comprised 0.5-μg total RNA, 1 × reverse transcription buffer (Toyobo Co., Ltd.), 1-mM deoxyribonucleotide triphosphate (dNTP) mixture (Toyobo Co., Ltd.), 5 units RNase inhibitor (Toyobo Co. Ltd.), 2.5 pmol oligo (dt) 20 (Toyobo Co., Ltd.), and 50 units ReverTra Ace. Reverse transcription was performed at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min using a programmable thermal controller. Finally, the cDNA samples were stored at −30°C until use.

RNA samples were extracted from the ileum and cecum using Sepazol RNA I Super (Nacalai Tesque Inc., Kyoto, Japan) according to the manufacturer's directions. RNA was dissolved in 10 mM Tris with 1 mM EDTA (pH 8.0) and stored at −80°C until use.
The RNA was treated with 1U RQ1 RNase-free DNase (Promega Co, Madison, WI, USA) in a 10-µl reaction mixture (10 µg total RNA, 1× DNase buffer and 1 U DNase) on a programmable thermal controller (PTC-100; MJ Research, Waltham, MA, USA) programmed at 37°C for 30 min and then at 65°C for 10 min. The reaction was stopped with 1U RQ1 DNase Stop Solution (Promega Corporation, Madison, USA). The concentration of RNA was measured using a NanoDrop Lite instrument (Thermo Fisher Scientific, Waltham, WV, USA). The RNA was then reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan) according to the manufacturer's instructions. The reaction mixture (10 µl) consisted of 0.5 µg total RNA, 1× reverse transcription buffer (Toyobo Co. Ltd.), 1 µM deoxyribonucleotide triphosphate (dNTP) mixture (Toyobo Co. Ltd.), 5 U RNase inhibitor (Toyobo Co. Ltd.), 0.25 µg of oligo(dT)20 (Toyobo Co. Ltd.), and 50 U ReverTra Ace. Reverse transcription was carried out at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min, in a programmable thermal controller (PTC-100; MJ Research). The cDNA samples were stored at −20°C until use.