Quantstudiotm 3

Total RNA was purified using TRIzol reagent (Invitrogen). RNA was reverse transcribed to cDNA using a PrimeScript RT kit (Takara, China). qRT-PCR was performed in QuantStudioTM3 (ThermoFisher) in a total reaction volume of 30 μL per well. The housekeeping gene ACT was used as the internal reference. All assays were performed in triplicate, and a reaction without reverse transcriptase was used as a negative control.

RT-PCR was used to quantify BDNF and TrkB expression. First, colon tissues were ground in liquid nitrogen for RNA extraction. For each sample, 50–100 mg tissue was extracted with 1 mL of RNA Trizol Reagent (Hefei Bomei Biotechnology Co., Ltd., China). The total RNA was used to synthesize cDNA. Primers (Shanghai SANGON Biotech Co., Ltd., China) are shown in Table 1. PCR reactions were performed on a real-time fluorescence quantitative instrument (QuantStudio TM3, Thermofisher, USA) with a total of 45 cycles. In this study, the relative expression levels of BDNF mRNA and TrkB mRNA were calculated by 2^−△△CT.

Total rat kidney RNA was extracted with RNA Trizol Reagent (Bomei, BM1144) and other reagents, and reverse transcribed into cDNA with PrimeScript RT reagent Kit (Takara Bio Inc., RR047A). Real-time polymerase chain reaction (RT-PCR) was performed using a QuantStudio TM3 instrument (Thermo Fisher Scientific) and CT (Threshold cycle) values of the PCR process were analyzed using Thermo Scientific PikoReal software. Comparative gene expression was calculated using the Livak method in this experiment. The following primers were used: MCP-1 (Monocyte Chemoattractant Protein-1) forward 5′-ctcacctgctgctactcattcactg-3′, reverse 5′-cttctttgggacacctgctgctg-3′; RNase6 forward 5′-agccatgcgtggtgtcaacaac-3′, reverse 5′-ggcagttcttccgaccgttcttg-3′; TNF-alpha (Tumor Necrosis Factor-Alpha) forward 5′-atgggctccctctcatcagttcc-3′, reverse 5′-cctccgcttggtggtttgctac-3′.

The qRT-PCR and melting curve analysis were performed using a QuantStudio^TM 3 real-time PCR (Thermo Fisher Scientific). mRNA levels were normalized by ACTB and compared to control (The first bar in each graph) [93 (link)] (Table S2).

Total RNA was extracted from cultured cells using the Qiagen Rneasy Mini kits. Complementary DNA was synthesized using a standard reverse transcription method (qScript cDNA SuperMix, Quanta Biosciences). qPCR reactions were performed using the SYBR Green PCR Master Mix (Thermo Fisher Scientific). The experiments were performed according to the manufacturer’s instructions using QuantStudioTM 3 Thermo Fisher Scientific Real-Time PCR system. The comparative CT method was used to determine relative gene expression levels for each target gene and 18S was used as an internal control for normalization (18S was the most stable gene among 4 reference genes tested). The sequences of the primers used for qPCR are available upon request.

Total RNA was extracted using the PureLink RNA kit (Life Technologies) according to the manufacturer’s protocol. cDNA was synthesised by reverse-transcribing 1 μg of RNA using the Tetro cDNA synthesis kit (Bioline) according to manufacturer’s protocol. To quantify gene expression using RT-qPCR, 2 μL of cDNA was added to a reaction mix containing: 10 μL 10x Power SYBR Green mix (Applied Biosystems, Life Technologies), 1 μL Forward primer, 1 μL Reverse primer, and 6uL of DEPC-treated H₂O. Differential gene expression was measured on the Quantstudio^TM 3 (ThermoFisher Scientific) using the standard protocol and data analysed using the log2ΔΔCt method. All mRNA expression levels were normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences (Supplementary Table 1) were obtained from Integrated DNA Technologies (NSW, Australia).