Dataassist software v3

Total RNA was isolated from mouse brown adipose tissue by using the RNeasy Lipid Tissue Mini Kit. iBAT tissue pieces of 10 mg from 5 mice from each group were pooled for total RNA extraction to represent wild type and Decr^−/− sample. RNA concentration and quality was analyzed using 2100 Bioanalyzer (Agilent Technologies). Total RNA from mouse liver was extracted by using using Versagene RNA tissue kit (Gentra Systems, Minneapolis, MN, USA). The RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) was used to produce cDNA from 1 μg of iBAT RNA or 500 ng of hepatic RNA. The 7500 Real Time PCR System was used with fluorogenic probe-based TaqMan chemistry and the relative standard curve method. TaqMan Gene Expression Assays (Supplementary Table S2) and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were used for iBAT samples throughout the qRT-PCR analyse. For hepatic FGF21, the primers and 5′FAM-labeled probe were designed using Primer Express software (Applied Biosystems) and the sequences available in Genbank and were purchased from Sigma-Genosys (Haverhill, UK). Mouse β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as endogenous controls to which sample values were normalized. Data was analyzed with DataAssist Software v3.0 (Applied Biosystems).

The results are expressed as the mean ± standard deviation (SD) or standard error of the mean (SEM). Statistical analyses were performed using the Statistical Analysis System (SAS Institute, Cary, NC). The non-parametric Kruskal-Wallis test was used between the different groups. Statistical significance was accepted at P<0.05. For the statistical analysise of gene expression, the DataAssist software V3.0 (Applied Biosystems, Life Technologies) was used to compare groups two by two through a two-tailed Student's t-test on the DeltaC_T values.

Total RNA was extracted from 10μm-thick sections of archival FFPE blocks using RecoverAll total nucleic acid isolation kit (Life Technologies, Grand Island, NY). The quality of RNA was assessed using the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Total RNA from breast cancer cell lines was isolated using miRNeasy kit (Qiagen, Germantown, MD). Total RNA was reverse-transcribed using the high capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. The mRNA level of DLC1 (Hs00183436_m1) and ECT2 (Hs00978168_m1) was analyzed by quantitative reverse transcription- PCR (qRT-PCR) using TaqMan gene expression assays on an ABI Prism 7900 platform (Thermo Fisher Scientific, Waltham, MA) with ACTB (Hs00357333_g1) and GUSB (Hs99999908_m1) as endogenous controls for normalization. All qRT-PCR reactions were performed in duplicates for tumor blocks and triplicates for breast cancer cell lines. The gene expression values was analyzed according to ∆∆Ct method using the Applied Biosystems DataAssist Software v3.0. GraphPad Prism 7.03 software was used to analyze statistical significance for qRT-PCR data.

For quantitative real-time PCR, RNAs were reverse-transcripted to cDNA using the Invitrogen SuperScript VILO™ Master Mix (No. 11755, Life Technologies). The expression of genes coding for known pro- and anti-angiogenic factors [25] (link), inflammatory factors, the glial cell marker and endogenous control genes (Table 1) was quantified using 5 ng of total cDNAs in 10 µl of 1X TaqMan Fast Advanced Master Mix (No. 4444557, Applied Biosystems, Life Technologies). Quantitative real time-PCR was performed on TaqMan Array 96-well FAST Plates (Applied Biosystems, Life Technologies), using the StepOnePlus Real-Time PCR System equipped with the StepOne software V2.2.2 (Applied Biosystems, Life Technologies). Data were analysed using DataAssist software V3.0 (Applied Biosystems, Life Technologies). Genes coding for glucuronidase-beta, beta-2-microglobulin and hypoxanthine guanine phosphoribosyl-transferease-1 were used as endogenous controls for normalization and relative quantification (RQ) with the Cycle Threshold (C_T)-method.

The statistical analysis was performed using R (version 3.4.0) to analyze the differences in mRNA and miRNA expression patterns in the MFS patients and HV controls. Raw data generated by the Agilent Feature Extraction image analysis software was normalized by variance stabilizing normalization (vsn) [16 (link)] and quantile normalization methods for mRNAs and miRNAs, respectively and uploaded to the NCBI GEO database (Accession ID: GSE110966). The significance level of mRNAs and miRNAs was determined by applying an unpaired two-tailed t test. Then the median values of each miRNA and mRNA were log2 transformed and the resulting miRNA P values were adjusted for multiple testing using Benjamini–Hochberg adjustment. In addition, the area under the receiver operating characteristic curve values for each miRNA were computed. For the significantly deregulated miRNAs and protein coding genes with P < 0.05 and fold change > 2 or < 1/2 in MFS patients compared to HV controls, we computed a Pearson correlation coefficient of expression for each mRNA–miRNA pair. Spearman’s correlations coefficient was used to correlate the clinical parameters of MFS and the expression level of both validated miRNAs and mRNAs. Using the DataAssist™ Software v3.0 (Applied Biosystems), the fold-change and P value (unpaired t test with Welch’s correction) of each mRNA and miRNA was calculated.

β actin, beta-2 microglobulin (B2M) and 18S were selected from a panel of reference genes using the geNorm component of the qBase 2.0 relative quantification model (Biogazelle, Belgium) as they were expressed stably across treatment groups (Control vs. MA) [36 (link)–38 (link)].
The mRNA expression of P-glycoprotein (P-gp) [36 (link)], CYP1A2, PXR, GR, 11βHSD1, 11βHSD2, MR, p50, p65, MCP1, SOD1, SOD2 and reference genes in liver samples were measured using KiCqStart SYBR Green qPCR ReadyMix Low Rox on a ViiA7 Fast Real-time PCR system (Applied Biosystems, California, USA) as previously described [35 (link), 36 (link)]. The reactions were quantified by setting the threshold within the exponential growth phase of the amplification curve and obtaining corresponding C_t values. DataAssist Software v3.0 (Applied Biosystems) [38 (link)] was used to find the 2^−ΔCt, which shows the abundance of each transcript relative to the abundance of the three stable reference genes and is expressed as mean normalized expression. DataAssist 3.0 analysis software (Applied Biosystems, California, USA) was used to normalise the abundance of target genes relative to the abundance of reference genes and expressed as mRNA mean normalised expression (MNE) ± SEM.

Total RNA was isolated using RNeasy plus mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, including treatment with RNase-free DNase I (Qiagen, Hilden, Germany). cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). qPCR was performed using qPCRBIO SyGreen Blue Mix (PCR Biosystems, London, UK) and run on a QuantStudio^TM 6 Flex Real-Time PCR System (Applied Biosystems). Primer sequences used were predesigned KiCqStart SYBR^® Green primers (Sigma-Aldrich Israel Ltd, Rehovot, Israel) and are included in Supplementary Materials Table S13. The expression of the indicated transcript was normalized to endogenous reference control GAPDH according to the ΔΔCt method using the DataAssist software v3.0 (Applied Biosystems).