Il 6 mm00446190 m1

Quantitative real-time polymerase chain reaction was performed as described previously [43 (link)]. The following primers were purchased from Thermo Fisher Scientific (Waltham, MA, USA): interleukin 1β (IL-1β; Mm00434228_m1), interleukin-6 (IL-6; Mm00446190_m1), tumor necrosis factor α (TNF-α; Mm00443258_m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Taq-Man Predeveloped Assay Reagents for gene expression, part number: 4352339E). GAPDH was used as an endogenous control.

Adipose tissue total RNA of eight samples from each group was isolated using RNeasy® Lipid Tissue Mini Kit Cat. #74104 (Qiagen, Manchester, UK). cDNA was synthesized using iScript Adv cDNA kit for RT-qPCR Cat. #1725038 (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.
For gene expression, a SYBR green-based, Cat. #172-5270 (Bio-Rad, Santa Rosa, CA, USA), real-time quantitative PCR (RT-qPCR) assay was performed on a Rotor Gene Q (Qiagen, Hilden, Germany) and relative expression ratios were determined by the Rotor-Gene Q Software 2.3.4.
Primers were designed online with the primer-BLAST tool and are listed in Table 1; GAPDH was used as a housekeeping gene. The procedure was performed with a 10 µl reaction mixture containing 5 ul of SYBR green enzyme, 0.4 ul of each forward and reverse primer, 2.2 ul of water, and 2 ul of cDNA under the following conditions: denaturation at 95 °C for 30 s, 45 cycles at 95 °C for 15 s, annealing temperature (varying for each gene as shown in Table 1) for 15 s, an extension of 72 °C for 30 s only for products longer than 150 pb, and the Melt ramp from 45 °C to 95 °C. Taqman probe was employed for IL-6 (Mm00446190_m1) (Thermo Fisher, Waltham, MA, USA).

RNA was extracted from cells using the Toolsmart RNA extractor (TB-DPT-BD24, Biotools), and then reverse-transcribed into complementary (c)DNA with a magic RT cDNA synthesis kit (Bio-Genesis Technologies, Taipei, Taiwan) according to the manufacturer's instructions. qPCRs were performed using TaqMan Gene Expression Master Mix (4369016, Thermo Fisher Scientific, Waltham, MA, USA) in an StepOnePlusTM machine (Thermo Fisher Scientific, Waltham, MA, USA). The TaqMan Gene Expression Assay used as follows: IL-6 (Mm00446190_m1), TNF-α (Mm00443260_g1), and IL-1β (Mm00434228_m1, was all from Thermo Fisher Scientific, Waltham, MA, USA). Results were normalized to the expression of the gene encoding 18s and were quantified by the change-in-threshold method (ΔΔCT).

qRT-PCR was carried out as previously detailed (22 (link)). RAW 264.7 cells using Trizol reagent (Thermo Fisher Scientific) and RNA easy mini kit (Qiagen, Germany). The quality and quantity of RNA were determined using the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). For cDNA synthesis, 2.0 ug of total RNA was reverse transcribed in a 50 ul reaction using Taqman reverse transcription reagents (Applied Biosystems, Foster City, Ca, USA) as per manufacturer’s instructions. Real time qRT-PCR was performed using QuantStudio™ 7 flex real-time PCR system with master mix reagents and premade TaqMan primers and probes for the following genes; TNF-α, [F: 5′-CCT CCC TCT CAT CAG TTC TAT-3′, R: 5′-CTA GTT GGT TGT CTT TGA GAT CC-3′, Probe: 5′-6-FAM-ACA AGC CTG TAG CCC ACG TCG TAG-BHQ-1-3′] (Metabion, Steinkirchen, Germany); Mm00443260_g1; IL-1β, Mm00434228_m1; IL-6, Mm00446190_m1; iNOS, Mm00440502_m1; CXCL2, Mm00436450_m1; CXCL10, Mm99999072_m1; and MCP-1 (CCL2), Mm00441242_m1(All from Applied Biosystems). The mRNA levels of target genes were normalized according to the comparative ΔCq method to respective mRNA levels of the housekeeping gene HPRT (Mm01545399_m1)(Applied Biosystems). The expression of the target gene is reported as the level of expression relative to HPRT.

Total RNA was extracted with NucleoSpin® RNA kits (Macherey-Nagel, Düren, Germany). RNA concentration was determined spectrophotometrically using NanoDrop® (ThermoFisher Scientific, Waltham, MA, USA). RNA was reverse transcribed with PrimeScript RT reagent kit using random hexamer primers (Takara, Kusatsu, Japan). TaqMan system was used for real-time PCR amplification. Relative gene expression was obtained after normalization to 18s RNA, using the formula 2^−ΔΔcp. The following primers/probes were used Il-6 Mm00446190_m1, Il-8 Mm00433859_m1, p16^Ink4a Mm00494449_m1 and p21 Mm00432448_m1 (Applied Biosystems, Rotkreuz, Switzerland).

We isolated RNA from brain tissue at indicated timepoints following tMCAO. Hemispheres were separated and homogenized in TRIzol Reagent (1 ml per 100 mg tissue), chloroform was added, samples were centrifuged at 12,000×g for 15 min at 4 °C and the upper aqueous phase was collected. RNA was precipitated by the addition of isopropyl alcohol, washed and dissolved in TE-Buffer. We isolated total RNA from cells using QIA-Shredder spin columns and the RNeasy Micro Kit (QIAGEN) and transcribed complementary DNA using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Fermentas). Real-time PCR primers were obtained from Applied Biosystems (Carlsbad, CA): Il6: Mm00446190_m1; Il23: Mm00518984_m1; Il1b: Mm00434228_m1; Tgfb: Mm01178820_m1; Cxcl1 Mm00433859_m1; Mmp3: Mm00442991_m1; BActin: Mm00607939_s1, Sdha: Mm01352366_m1. Probe mixtures were purchased from Fermentas (Waltham, MA). The relative gene expression was calculated using the ΔΔCt method and the samples were normalized to the control population and the expression of Sdha or β-Actin. Samples were randomized and coded by an independent researcher, so experiments were carried out blindly.