Quantitec rt kit

Rat’s hearts were washed in PBS, and immediately separated from risk and remote zones. Each zone was cut in small pieces collected in RNA later (Qiagen N.V., Germany), and processed according to the extraction of RNA. Total RNA was extracted from tissues by homogenization using tissuelyzer (Qiagen N.V., Germany) with 1 ml of Trizol. After chloroform mix and centrifugation, aqueous phase was transferred to “RNeasy mini kit” (Qiagen N.V., Germany) columns and were continued according to the manufacturer’s instructions. The ratio of absorbance at 260 and 280 nm (A260/A280) was used to assess the purity of RNA. RNA concentrations were determined from absorbance at 260 nm (A260). Two microgram of RNA were retrotranscribed to cDNA with the “RT quantitec kit” (Qiagen N.V., Germany). Standard qRT-PCRs were performed as described previously (Díaz et al., 2017 (link)). Data analysis was made with the “Expression Suite” software and fold change quantification was calculated using the comparative cycle threshold CT (ΔΔCT) method. 18s rRNA gene was used as endogenous control.

Total RNA was extracted from cultured cardiac myocytes using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. RNA purity and concentration were assessed by measuring absorption at 260 nm and 280 nm. 1 μg of RNA were retro-transcribed to cDNA with the RT quantitec kit (Qiagen). qRT-PCRs were performed with the use of a Prism 7900HT Sequence Detection System, Taqman primers and probes technologies (Applied Biosystems). Fold change in gene expression was calculated using the comparative cycle threshold CT (ΔΔCT) method.
For apoptosis PCR array, we used plates from SA Biosciences (Cat. PAHS-012). Total RNA from Ucn-1 treated and no-treated cardiac myocytes (as described in cell treatment protocols) submitted to I/R was extracted and reverse transcribed into cDNA with the use of an RT2 First Strand Kit (SA Biosciences). The templates were combined with an RT2 SYBR Green qPCR Master Mix (SA Biosciences), and then equal aliquots of this mixture (25 μl) were added to each well of the same PCR Array plate that contained the pre-dispensed gene-specific primer sets. The qRT-PCR quantification was performed as described above by ΔΔCT method.

The RNA was isolated from unstimulated, LPS-stimulated neutrophils in the presence or absence of phloretin, CAMKII, Apoc, and IL-10 using the RNAeasy mini kit (Qiagen). cDNA samples for mRNA expression were synthesized using the Quantitec RT kit (Qiagen) according to our previous reports [57 (link),58 (link)]. For quantitative real-time PCR, samples were run in duplicate using Quantitect LGALS9 primers (QT02309545), and Beta-2-microglobulin (QT01149547) was used as a reference gene (Qiagen). Samples were analyzed by the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Data analysis was carried out using the 2^−ddCT method.