Iscript cdna synthesis kit rt

Total mRNA extraction was obtained with the QIAzol Lysis Reagent (Qiagen Sciences, Maryland, USA); integrity and quantification of each sample was determined with a NanoDrop ND‐1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). cDNA synthesis was performed using iScript™ cDNA synthesis kit RT (Bio‐Rad Laboratories Inc., Hercules, California, USA), starting from 500 ng of mRNA. Primers for Agtn, angiotensin‐converting enzyme (Ace), angiotensin type 1 receptor (At1r), neprilysin (Nepr), Tlr4, monocyte chemoattractant protein‐1 (Mcp1), collagen type I (Coll1), Tnfα, Mstn, Nfe2l2 (NRF2), NAD(P)H dehydrogenase [quinone] 1 (Nqo1), and β‐actin were obtained from Tib Molbiol Srl (Genova, Italy). The sequences are reported in Table 1. PCR amplification was carried out using the probe SYBR Master Mix solution (Eppendorf, Hamburg, Germany) on a MasterCycler RealPlex (Eppendorf) PCR system. Βeta‐actin was chosen as reference gene; gene expression was calculated with the ∆∆CT method of relative quantification and expressed as fold change vs. CTR cells.

cDNA synthesis was performed using the iScript cDNA synthesis kit RT (Bio-Rad Laboratories Inc., Hercules, CA, USA). Primers were obtained from Tib Molbiol S.r.l. (Genoa, Italy) and sequences are reported in Table 1. PCR amplification was carried out in a total volume of 10 μL, containing 1 μL of cDNA solution, 5 μL of SYBR Master Mix solution (Eppendorf, Hamburg, Germany), 0.25 μM of each primer, and nuclease-free water. Based on their stability in respect to treatments, β-actin (for exposure to ischemia) or β2-microglobulin (for exposure to reperfusion) were quantified and used to normalize the expression of the other genes. The ∆∆CT method of relative quantification was used to determine the fold change in expression. Assays were run in triplicate using a Universal PCR Master Mix on a MasterCycler RealPlex PCR system (Eppendorf, Hamburg, Germany).

cDNA synthesis was performed using the iScript™ cDNA synthesis kit RT (Bio-Rad Laboratories Inc., Hercules, California, USA). MCP1, TGF-β, collagen I, NOX4, and GAPDH primers were obtained from Tib Molbiol Srl (Genoa, Italy), and sequences are reported in Table 1. PCR amplification was carried out in a total volume of 10 μl, containing 1 μl of cDNA solution, 5 μl of SYBR Master Mix solution (Eppendorf, Hamburg, Germany), 0.03 μl of each primer, and 3.94 μl of nuclease-free water. GAPDH was quantified and used for the normalization of expression values of the other genes. Fluorescence signals measured during the amplification were considered positive if the fluorescence intensity was more than 20-fold greater than the standard deviation of the baseline fluorescence. The ∆∆CT method of relative quantification was used to determine the fold change in expression. Assays were run in triplicate using Universal PCR Master Mix on a MasterCycler RealPlex (Eppendorf) PCR system.