Lightcycler software 3

For RNA extraction the NucleoSpin RNA Kit (MACHEREY-NAGEL) was used. Total RNA concentration was determined photometrically using Nanodrop (Peqlab), and RNA quality was assessed by electrophoresis on a denaturing agarose gel. Using the SuperScript III Reverse Transcriptase (Invitrogen), 1 μg of total RNA was reverse transcribed. Quantitative RT-PCR analysis was carried out on the LightCycler 2.0 Instrument (Roche) using the Mastermix LightCycler FastStart DNA Master SYBR Green I (Roche). Samples were analyzed in duplicate and averaged using the Light Cycler Software 3.5 (Roche). Data were analyzed based on the ΔΔCt method using HT29 Diaph2 control cells as a reference sample. All primers (Table S1) were design to amplify products between 90 and 200 bp, and exon-spanning primers were used to avoid DNA amplification.

RNA from the liver was isolated with the RNeasy Mini Kit (Qiagen, MA, USA). mRNA expression levels of Tnf-α, Il6, Mcp1, Toll-like receptor 2 (Tlr2), Toll-like receptor 4 (Tlr4), peroxisome proliferator-activated receptor gamma (Ppar-γ), and β-actin (Actb) were measured by qRT-PCR (for primer sequences used in the study see Table S2.) qRT-PCRs were carried out on LightCycler 1.5 (Roche, Mannheim, Germany) with the Light Cycler TaqMan Master Kit (Roche, Mannheim, Germany). Preincubation was performed at 94 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 10 s, then annealing at 60 °C for 30 s, and extension at 72 °C for 1 s. LightCycler^® Software 3.5 (Roche, Mannheim, Germany) was used to perform relative quantification, with β-actin as internal control.

Total RNA extraction from MECs harvested at CTR, 30m, 2h, 4h, 8h, 12h, 24h after heat stress and cDNA synthesis was performed as described in our previous studies [13 ]. Primer details for all the heat shock protein and apoptotic gene family are provided as supplementary information (S1 Table). The accuracy of primer pairs was also ensured by the presence of a unique peak during the dissociation step at the end of qPCR cycle. qPCR was performed using Light Cycler 480 instrument (Roche, Germany) as described in our previous reports [13 ]. The data was acquired using the ‘second derivative maximum’ method as computed by the Light Cycler Software 3.5 (Roche Diagnostics) and subjected for subsequent analysis.

RNAs were extracted with the Nucleospin^® RNA extraction kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. Reverse transcription of RNA was performed with the Transcriptase inverse SuperScript II (Invitrogen, 18064022), following the manufacturer’s instructions. Quantitative PCRs were performed with 1 μL of 1/10 diluted first strands of cDNA in a total volume of 15 μL of 1X KAPA SYBR FAST qPCR Master Mix (Kapa Biosystem, KK4610, Merck, Darmstadt, Germany), containing 10 nM of each PCR primer (Table 2). Reactions were performed in 20 μL LightCycler capillaries (Roche). PCR amplifications were performed with a LightCycler 1.5 thermocycler and analyzed with the LightCycler Software 3.5 (Roche, Basel, Switzerland). Assays were performed in technical duplicates on RNAs extracted from two or three independent experiments.

Blood samples were collected by venipuncture into EDTA tubes using the Vacutainer system. DNA was extracted from leukocytes using a commercial automated procedure (Roche, Italy). The DNA was spectrophotometrically quantified (also to verify the purity) and analyzed for FVL (R506Q, rs6025); FVR2 (H1299R, rs1800595); FII (G20210A, rs1799963); MTHFR (C677T, rs1801133 and A1298C, rs1801131); beta-fibrinogen (-455 G>A, rs1800790); FXIII (V34L, rs5985); HPA-1 (L33P, rs5918) and PAI-1 (4G/5G alleles, rs1799889). All the variants were analyzed using a LightCycler 1.2 Instrument, which uses PCR for the amplification of the genomic region of interest and fluorogenic target-specific hybridization for the detection and genotyping of the amplified DNA, according to the manufacturer’s procedures (Roche Diagnostics). Finally, the results were analyzed using LightCycler Software 3.5. (Roche Diagnostics, Basel, Switzerland).

Blood samples were collected by venipuncture into EDTA tubes using the Vacutainer system. DNA was extracted from leukocytes using a commercial automated procedure (Roche, Italy). The DNA was spectrophotometrically quantified (also to verify the purity) and analyzed for FVL (R506Q variant); FVR2 (H1299R variant); FII, G20210A; MTHFR, C677T and A1298C; beta-fibrinogen, -455 G>A; FXIII, V34L; HPA-1, L33P variants and PAI-1 4G/5G alleles. All the variants were analyzed using a LightCycler 1.2 Instrument, (Roche Diagnostics, Basel, Switzerland), which uses PCR for the amplification of the genomic region of interest and fluorogenic target-specific hybridization for the detection and genotyping of the amplified DNA, according to the manufacturer’s procedures (Roche Diagnostics). Finally, the results were analyzed using LightCycler Software 3.5 (Roche Diagnostics, Basel, Switzerland) [18 (link)].

After sacrifice, a portion of the muscle was prepared in order to first extract the total cellular RNA using RNeasy Mini Kit (Qiagen, Waltham, MA, United States) in accordance with the manufacturer’s instructions and then to reverse transcribe the material into complimentary DNA(cDNA) using MMLV reverse transcriptase (Promega, Madison, WI, United States). The expression of glucose transporter 1 (GLUT1), glucose transporter 4 (GLUT4), and β-actin were quantified using qRT-PCR. Respective primer sets (forward and reverse) referred to previous study (Hu et al., 2014 (link)) as follows: GLUT4, 5-TGCTCTCCT GCAGCTGATT-3, and 5-TTCAGCTCAGCTAGTGCGTC-3; GLUT1, 5-CTTCCTGCTCATCAATCGT-3, and 5-AGCTCCA AGATGGTGACCTT-3; and Beta-actin, 5-CTAAGGCCAACC GTGAAAAG-3, and 5-ACCAGAGGCATACAGGGACA-3. Levels of GLUT4 and GLUT1 were normalized against the amount of Beta-actin mRNA. Reactions were performed on a LightCycler 1.5 using a LightCycler TaqMan master kit (both from Roche, Mannheim, Germany). Preincubation was performed at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 1 s, and cooling at 40°C for 30 s. LightCycler^® Software 3.5 (Roche) was used to perform relative quantification using beta-actin as an internal control.