Takara rt kit

Total RNA was extracted from cardiac tissues and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The RNA was reverse transcribed into cDNA using the Takara RT kit (Takara Biotechnology Co., Ltd.) at 37°C for 15 min. The expression levels of miR-26b, collagen I, α-smooth muscle actin (α-SMA), Keap1 and Nrf2 were determined using the SYBR Green PCR kit (Takara Biotechnology Co., Ltd.). GAPDH and U6 were used as controls. The following amplification conditions were used: Pre-denaturation at 95°C for 15 sec, denaturation at 94°C for 30 sec, annealing at 60°C for 20 sec, and extension at 72°C for 40 sec for 40 cycles. Relative gene expression was calculated using the 2^−ΔΔCq method (25 (link)). The sequences of the primers were as follows: miR-26b forward, 5′-CCCAGTTCAAGTAATTCAGG-3′; and reverse, 5′-TTTGGCACTAGCACATT-3′; collagen I forward, 5′-CAGAGCACGATGTCCTGAGA-3′; and reverse, 5′-GCAAATGTGAGCTTCTGTGC-3′; α-SMA forward, 5′-GGAGTGATGGTTGGAATGG-3′; and reverse, 5′-ATGATGCCGTGTTCTATCG-3′; Keap1 forward, 5′-GGACGGCAACACTGATTC-3′; and reverse, 5′-TCGTCTCGATCTGGCTCATA-3′; Nrf2 forward, 5′-CACATCCAGACAGACACCAGT-3′; and reverse, 5′-CTACAAATGGGAATGTCTCTGC-3′; GAPDH forward, 5′-CAAGCTCATTTCCTGGTATGAC-3′; and reverse, 5′-CAGTGAGGGTCTCTCTCTTCCT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACATATACTA-3′; and reverse, 5′-ACGAATTTGCGTGTCATCCTTGCG-3′.

Total RNA was extracted from SW-13 cells cultured in different concentrations of TUDCA (0, 100, 200, 300, or 400 µM for 48 h) using RNAiso Plus reagent (Takara Biotechnology Co., Ltd.). Following RNA extraction, cDNA was synthesized using a Takara RT kit (Takara Biotechnology Co., Ltd.) at 37°C for 15 min, 85°C for 5 sec and kept at 4°C until further experimentation. qPCR amplification was conducted with SYBR-Green reagent (Takara Biotechnology Co., Ltd.) using an ABI 7500 sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the following conditions were used: Initial denaturation at 95°C for 30 sec, then 40 cycles of 95°C for 5 sec and 60°C for 34 sec. The experimental steps were carried out according to the manufacturer's instructions. The primers were designed and synthesized by Sangon Biotech Co., Ltd. (Table I). C_q values were generated using the default analysis settings. ΔC_q was calculated using the following formula: (C_q gene of interest)-(C_T GAPDH). ∆∆C_q was calculated using the following formula: (ΔC_q treated sample)-(C_q control sample). Relative expression was calculated using the 2^−ΔΔCq method as previously described (20 (link)).

Total RNA was extracted from cells using Total RNA isolation reagent (Biosharp) and then reverse-transcribed into cDNA using a Takara RT kit (Takara Bio, Inc.). The mixture was incubated at 37°C for 15 min, followed by 85°C for 5 sec and then cooled at 4°C. qPCR was performed using TB Green^® Premix Ex Taq™ (Takara Bio, Inc.) in a final volume of 20 µl in an ABI 7500 reactor (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR conditions consisted of an initial denaturation step for 30 sec at 95°C, followed by 40 cycles of 3 sec denaturation at 95°C and annealing/elongation for 30 sec at 60°C. The samples were quantified using the 2^−ΔΔCq method and β-actin was used as the internal control (25 (link)). The PCR primer sequences (Sangon Biotech, Co., Ltd.) are listed in Table I.

Total RNAs of the treated KHOS and U2OS cells were prepared using the TRIzol reagent (Takara, Japan) and then transcribed into cDNA using a Takara RT kit (Takara) following protocols obtained from the manufacturers. The ABI Prism 7700 sequence detection system (PE Applied Biosystems, California, USA) was used to carry out the quantitative real-time polymerase chain reaction process with the following protocols: 95 °C, 5 mins; 35 cycles of 95 °C for 35 s, 60 °C for 45 s, and 72 °C for 90 s; and 72 °C, 10 mins. Sequences of primers used in the present study are the following: Sox2: forward 5′-CAC CTA CAG CAT GTC CTA CTC G-3′, reverse 5′-GGT TTT CTC CAT GCT GTT TCT T-3’; Oct4: forward 5′- GCT CGA GAA GGA TGT GGT CC-3′, reverse 5′- CGT TGT GCA TAG TCG CTG CT-3’; Nanog: forward 5′-GGA GTA GAG TGT AGA GGA GAA TGA GTT A-3′, reverse 5′-CTA ACT CTT TAA CTT CTT CCC AAA TC-3’; GAPDH: forward 5′- TGT TCG TCA TGG GTG TGA AC-3′, reverse 5′- ATG GCA TGG ACT GTG GTC AT-3’; GAPDH was used as the internal control, and the expression of each gene was calculated using the 2^−ΔΔCT method.