Kicqstart sybr green primers

Cells were seeded in 60 mm dishes and cultivated overnight, and total RNA was isolated with TRI Reagent^® (Merck) and a Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA, USA). cDNA synthesis was performed with 1 µg of isolated RNA using a GoScript™ Reverse Transcriptase kit (Promega, Fitchburg, WI, USA). Quantitative real-time PCR was performed on a ViiA7 real-time PCR system (Applied Biosystems, ThermoFisher Scientific) using GoTaq^® Master Mix (SYBR^® Green, Promega). mRNA expression levels of HDACs are presented as ΔCt values related to beta-actin. Primers were purchased as 100 µM stocks (KiCqStart^® SYBR^® Green Primers, Merck). Primer sequences are listed in Figure S1.

Expression analysis of NKA subunits was done via measurement of mRNA levels. In short, cells were seeded in 60 mm dishes and let grown for 24 hours. Cells were washed with PBS and harvested using Tri Reagent® (Merck). RNA was isolated via a Direct-zol™ RNA MiniPrep (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. cDNA was generated using a GoScript™ Reverse Transcriptase Kit (Promega). Quantitative real-time PCR was performed on a ViiA7 real-time PCR system (Applied Biosystems, ThermoFisherScientific) using GoTaq® Master Mix (SYBR® Green, Promega) using the following protocol: initial denaturation (95°C, 2 minutes) followed by 45 cycles (95°C for 3 seconds and 60°C for 30 seconds). Each biological replicate contained four technical replicates. A melting curve analysis was performed to guarantee the specificity of the products. mRNA expression levels of NKA subunits are presented as ΔCt values related to beta-actin. Primers were purchased as 100 μM stocks (KiCqStart® SYBR® Green Primers, Merck). Primer sequences are listed in S1 Fig.

Total cellular RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany). RNA concentration and purity were assessed using NanoDrop ND-1000, (Thermo Fischer Scientific, Wilmington, DE). RNA was transcribed into cDNA using the AffinityScript QPCR cDNA Synthesis Kit and oligo(dT) primers according to the manufacturer’s protocol (Agilent Technologies, Santa Clara, CA, USA). Real-time measurement of mRNA levels was performed with Stratagene 3005P qPCR System (Agilent Technologies) using TaqMan^® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) specific for each gene of interest (GOI; see Supplementary Table S1 for the list of the assays), apart from STING and CGAS, which were detected by SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) with specific KiCqStart^® SYBR^® Green Primers (Merck, Darmstadt, Germany) (see Supplementary Table S1). Glyceraldehyde 3-phosphate dehydrogenase (GADPH) expression was used as a normalization control, and results were expressed as 2^(−ΔCt), where ΔCt = Ct GOI − Ct GAPDH.

RNA was extracted from liver, muscle and BAT tissue using 1 mL TRIzol added to samples prior to homogenisation within a mechanical homogeniser for 2–3 min. Chloroform was added to each individual sample prior to gentle shaking and centrifugation at 16000 × g for 15 min. The supernatant was decanted into separate Eppendorf tubes and a 1:1 mixture of supernatant to 70% iso-propanol was made prior to further centrifugation at 16000 × g for 15 min. Following this the RNA pellet was washed three times with 70% ethanol and resuspended in sterile RNase-free water. RNA was quantified in addition to quality assessed using a NanoDrop spectrophotometer and 1 μg of RNA was then used for the synthesis of cDNA using the iScript cDNA synthesis Kit (Bio-Rad). Quantitative polymerase chain reaction (qPCR) was then undertaken using the SYBR-Green method using KiCqStart SYBR Green primers (Merck).

500 ng of total RNA prepared for microarray (as above) was reverse–transcribed using superscript III Reverse transcriptase (Life technologies) according to manufacturer’s instructions. RT-qPCR was carried out on a Stratagene Mx3005P instrument (Agilent Technologies) using either RT² SYBR^® Green qPCR mastermix (Qiagen) and RT² qPCR primer assays for Gapdh, Tlr1, Cd40, Tnf, Lamp2, and Ctsl or Platinum SYBR^® Green qPCR SuperMix-UDG (Invitrogen, USA) and KiCqStart™ SYBR^® Green primers (Sigma) for Cd163 and Lcn2 as detailed in Tables 1 and 2, respectively, according to manufacturer’s instructions. qPCR cycles were performed as follows: hot-start denaturation cycle 95°C for 10 min, 40 cycles of amplification of 95°C for 15 s and 60°C for 1 min ΔCt (Ct of housekeeping gene, Gapdh, minus Ct of gene of interest) was calculated for each sample. Data are expressed as the normalized expression ratio (2^(−ΔΔCt)) in comparison to sham samples.

The total RNA of cultured cybrid cells treated with PU-91, quercetin, (PU-91+quercetin), and DMSO was isolated. The information of all target primers, which are predesigned via Qiagen QuantiTect Primer Assays or KiCqStart SYBR^® Green primers (Sigma–Aldrich, Burlington, MA, USA), is demonstrated in Supplementary Table S1. The qRT-PCR was performed to assess the relative expression levels of genes associated with apoptosis (BAX and CASP3), inflammation (IL6), mitochondrial biogenesis regulators (TFAM, NRF1, and PGC1α), antioxidant enzyme (SOD2), and senescence (CDKN1A and LMNB1) pathways. HPRT1, a recycling enzyme of inosine and guanine in the purine salvage pathway, was selected as the housekeeping gene. Therefore, as a stable endogenous control gene, the HPRT1 primer was considered the reference gene for reaching standard expression levels for all primers. For analyzing the obtained data, the ΔΔCt method was used, in which ΔCt = [Ct (threshold value) of the target gene] − [Ct for HPRT1]; and ΔΔCt = ΔCt of the treatment condition − ΔCt of the untreated condition. For the comparison between untreated conditions versus treated conditions, the fold changes were measured as follows: fold change = 2^−ΔΔCt. Triplicate formats of treated cells (PU-91, quercetin, and PU-91+quercetin) compared to vehicle-control (DMSO) samples were analyzed.