Real time pcr instrument

For quantitative real time PCR, fresh frozen interventricular septum samples of patients with end stage HF were analyzed. Total RNA was isolated with the RNeasy Mini kit (Qiagen, Venlo, Netherlands, 74104) following homogenization of all tissue samples in a Precellys^® Evolution homogenizer equipped with a Cryolys^® dry ice cooling system (Bertin Technologies, Montigny-le-Bretonneux, France). Experimental procedure strictly followed product catalog guides. RNA concentrations were measured by spectrophotometry in NanoDrop (Thermo Fisher Scientific, Waltham, MA, United States, ND2000). cDNA was reverse transcribed from extracted RNA using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814). TaqMan Gene Expression Assays were used to quantify the mRNA expression levels of genes of interests: Relaxin-1 (Hs04194320_s1), Notch-1 (Hs01062014_m1), and ACTA-2 (Hs00426835_g1). Human glyceraldehyde 3-phosphate dehydrogenase GAPDH (Hs02758991_g1) was used as endogenous housekeeping gene. The PCR was performed with real-time PCR instrument (Applied Biosystems, Foster City, CA, United States, StepOnePlus) and the relative expressions were determined by ΔΔCt method.

Trizol reagent (Cat.: 15596026, Life Technologies, MA, USA) was applied to isolate the total RNA from tissues or cultured cells in accordance with the manufacturer’s instructions. Superscript III reverse transcription kit was used to obtain 20 ug/L RNA with a final volume of 10μl (Cat.: 18080200, Life Technologies). Real-time PCR was performed with gene-specific primers in the presence of SYBR Premix Ex Taq (Cat.: RR420A, TaKaRa, Japan). qPCR amplification conditions were as follows: 95°C for 10 min, 94°C for 30 s, 60°C for 15 s, and 72°C for 30 s for 35 cycles in Real-Time PCR Instrument (Applied Biosystems, USA). Relative mRNA expression level was calculated using the formula2-^ΔΔCt.

Thermal stability scans of rhGAA were performed as described in Porto et al (2012 (link)). Briefly, the enzyme was diluted 48‐fold (0.1 mg/ml in 25 mM Na‐phosphate buffer, pH 7.4, 150 mM NaCl, and 1:1,000 SYPRO Orange dye (Life Technologies)). Thermal scans were performed in triplicate in absence or in presence of 1 mM, 5 mM, and 10 mM antioxidants, with steps of 1°C per minute in the range 25–95°C in a Applied Biosystems Real‐Time PCR Instrument. Fluorescence was normalized to the maximum value within each scan to obtain relative fluorescence. Melting temperatures were calculated according to Niesen et al (2007 (link)). The standard deviations for each melting temperature were calculated from three replicates.
The dissociation constant (K_D) of Edaravone was measured by thermal stability scans of rhGAA according to (Vivoli et al, 2014 ). DSF scans were performed as described above, in the range 0‐18 mM Edaravone. The melting temperature values were plotted as function of ligand concentration. Experimental data were best fitted according to a simple cooperative model equation reported in Vivoli et al (2014 ) by using the software GraphPad Prism (GraphPad Software, San Diego, California, USA).

We collected yeast cells grown for 6 h in batch fermentation and used them for RNA preparation and Real-Time Quantitative PCR (RT-qPCR) Assay. The preparation of yeast RNA was performed by AccuPrep^® Universal RNA Extraction Kit from BIONEER (Daejeon, Korea). The detailed experimental methods followed the AccuPrep^® Universal RNA Extraction Kit Protocol. The cDNA synthesis of yeast RNA was carried out using the PrimeScript^™ 1st strand cDNA Synthesis Kit from Takara Bio (Shiga, Japan) and its protocol.
The RT-qPCR was performed to determine the RNA expression using a Real-Time PCR instrument (Applied Biosystems, CA, USA). The parameters for PCR were as follows: pre-incubation at 95 °C for 30 s, then 40–50 cycles of amplification at 95 °C for 5 s, 55 °C for 20 s, and cooling at 50 °C for 30 s. The relative expression of the CAR1, DUR1, 2, and DUR3 genes was determined by the ΔΔCt method based on the ACT1 gene which is known to be continuously expressed [17 (link),18 (link)]. Primers used for RT-qPCR analysis are summarized in Table 2.