Mir x mirna first strand synthesis rt kit

The cDNA used for the detection of miRNA expression was synthesized from 500 ng of high-purity total RNA obtained by phenol-chloroform extraction method. For this purpose, the cells’ pellets were homogenized with 0.5 mL of trizol (TRI Reagent^®). The RNA isolation procedure was performed according to the protocol supplied by the manufacturer. The Mir-X miRNA First-Strand Synthesis RT Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to obtain cDNA in the reverse transcriptase reaction. The quantity of miRNA transcript was investigated with real-time PCR technique. The total volume of PCR reaction was 20 μL. The reaction was performed using 0.5 μL of template, and the final concentration of primers was 0.4 μM. The following cycling conditions were applied during the reaction: a temperature of 95 °C for 10 s, followed by 35 cycles at a temperature of 95 °C for 5 s and annealing temperature of 57.4 °C for 20 s with a single fluorescence measurement. The list of miR specific primers used in the reaction is presented in the Table 2. The mRQ 3′ primer and U6snRNA primers were provided with the RT kit. The average fold change in the gene expression of experimental cultures was compared with control cultures and calculated by the 2−DDCt method in relation to U6snRNA.

Total RNA was used to generate cDNA using a Mir‐X miRNA First‐Strand Synthesis RT Kit (Clontech Laboratories, Inc) according to manufacturer's recommendations. The cycle parameters for the RT reaction were 30°C for 10 minutes, 55°C for 5 minutes, 37°C for 1 hour and 85°C for 5 minutes. Subsequently, the synthesized cDNA was used for real‐time quantitative PCR with SensiFAST SYBR Green Kit (Bioline). The reaction was performed using 0.5 μL of template, and the final concentration of primers (Table 2) was 0.4 μmol/L. The following cycling conditions were applied during the reaction: a temperature of 95°C for 10 seconds, followed by 35 cycles at a temperature of 95°C for 5 seconds and annealing temperature of 58.8°C for 20 seconds with a single fluorescence measurement. The mRQ 3′ primer and U6snRNA primers were provided with the RT kit. The average fold change in the gene expression of experimental cultures was compared with control cultures and calculated by the 2 − DDCt method in relation to U6snRNA.