Taqman primers and probes technologies

Total RNA was extracted from cultured cardiac myocytes using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 1 µg of RNA were retro-transcribed to cDNA with the TaqMan microRNA Reverse Transcription Kit (Qiagen, Hilden, Germany). qRT-PCRs were performed using a Prism 7900HT Sequence Detection System, TaqMan primers and probes technologies (Applied Biosystems, California, USA). Fold change in gene expression was calculated using the comparative cycle threshold CT method (2^−ΔΔCT method) using U87 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.