A3500

Real-time PCR was carried out as described previously^{38 (link),39 (link)}. Total RNA was purified from brain tissue using TRIzol reagent (15596018, Invitrogen Life Technologies, Carlsbad, CA, USA) and was treated with DNase I (M610A, Promega, MO, USA) to eliminate genomic DNA contamination. Reverse transcription of total RNA (2 μg) was conducted in a volume of 20 μl using a reverse transcription system (A3500, Promega, MO, USA). Real-time PCR was performed with 10 μl of 2X iQ™ SYBR Green Supermix (#170882, Bio-Rad Laboratories, Foster City, CA, USA), 1 μl each of 5 pmol/μl forward and reverse primers, and 4 μl of complementary DNA (cDNA) (1/8 dilution of the conversion) in a total volume of 20 μl using a CFX 96 Real-Time PCR System Detector (Bio-Rad Laboratories). The Bio-Rad CFX Manager 3.1 was used to analyze qPCR data. The primer sets used in PCR were listed in Supplementary Data 5.

TRIzol Reagent (15596‐018; Invitrogen) was used for total RNA extraction, and then total RNA was reverse‐transcribed into cDNA with reverse transcription system (A3500; Promega). Quantitative real‐time reverse transcription polymerase chain reaction (qRT‐PCR) was performed using SYBR Premix Ex Taq kit (RR420A; TaKaRa) on a Stratagene Mx3000P quantitative PCR system (Genetimes Technology). The PCR program was 95°C, 10 minutes; 95°C, 15 seconds, 60°C, 1 minute, 40 cycles. There were three technical repetitions for all the reactions. The primers used in the study are listed in Table S1.

Total RNA was extracted from cell lines using Trizol reagent according to the manufacturer’s instructions (Invitrogen^®). The quantity and quality of the isolated RNA were checked using NanoDrop™ One Spectrophotometer (Thermo Scientific^® [Asnières sur Seine, France]). Reverse transcription of 1 mg of total RNA into first strand cDNA was done using the reverse transcription system (A3500, Promega^® [Charbonnières-les-Bains, France]). Fast SYBR Green master mix (Applied Biosystems^® [Asnières sur Seine, France]) was used to perform the real-time quantitative PCR on a StepOnePlus system (Applied Biosystems^®). The relative expression was calculated using the ∆Ct method and RPLP0 mRNA level was used as the endogenous control to normalize relative expression values of each target genes. Gene-specific primer sequences are summarized in the Table S2 in Supplementary Materials.

Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Germany, 74,034) according to the manufacturer’s protocol. Then, RNA concentration was determined by NanoDrop 2000 (Thermo Fisher) and cDNA was obtained from total RNA (50 ng) of each sample by reverse transcription using oligo (dT)₁₅ and reverse transcriptase (Promega, USA, A3500), according to the manufacturer’s instructions, at 42 °C for 15 min, followed by 95 °C for 5 min in 20 μl total volume.
Quantitative real-time PCR was performed using SYBR Green Master Mix (ABI, Germany, DBI-2044) in an Applied Biosystems 7500 Real-Time PCR System. An aliquot (10%) of cDNA was subjected to 40 amplification cycles of PCR with the primers listed in Supplementary Table 1. Cycling parameters were one cycle for 2 min at 50 °C, one cycle for 10 min at 95 °C, 40 cycles at 95 °C for 15 s, and 60 °C for 20 s. For each experiment, three replicates were included in each qPCR reaction. We performed melting curve analysis at the end of each run to ensure a single amplicon.
The relative levels of endogenous β-actin mRNA (ACTB) mRNA were used as an internal control [40 (link)], and data were analyzed using the 2^-(△△Ct) method. Only samples whose cycle threshold (Ct) values of ACTB were between 21 and 22 were included in the analysis.

Total RNA was extracted from wild type and MIF knockout mouse bladder tissue through Trizol (15596026, ThermoFisher Scientific, Grand Island, NY), DNA removed by DNase, and reversed transcribed to cDNA (A3500, Promega, Madison, WI). SYBR green (4472903, ThermoFisher Scientific, Grand Island, NY) was utilized with primers (HMGB1, PPM05059F; Rn18s, PPM72041A, Qiagen, Germantown, MD) to quantified level of mRNA in bladder tissue from wild type and MIF knockout mice. 18S rRNA was used as the internal control.