Biomaster hs qpcr sybr blue master mix

Total RNA from leukemic cells was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA quantification was performed with SYBR Green-based real-time qPCR using IQ5 Cycler (Bio-Rad, Hercules, CA, USA). The cDNA was amplified in a total volume of 20 µL containing 2 µL of cDNA template, RNA specific primers (1 µM), and BioMaster HS-qPCR SYBR Blue master mix with SYBR Green I fluorescent dye (Biolabmix, Novosibirsk, Russia). The following primers were used: MDR1_F: 5’-AGAGAATCCCCTCCAGATAAGA–3’ and MDR1_R: 5’-AAGCCTATTCCATTTTGAACTTTCT-3’, GAPDH_F: 5’-GTGAAGGTCGGAGTCAAC-3’ and GAPDH_R: 5’-TGGAATTTGCCATGGGTG-3’. All measurements were done in triplicate. The amount of RNA was calculated from the number of cycles using standard curves and the results were normalized to glyceraldehyde 3 phosphate dehydrogenase (GAPDH). The obtained PCR data were analyzed using Bio-Rad iQ5 v.2.0 software.

Total RNA was extracted from tumor cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quantification was performed with real-time qPCR using BioMaster HS-qPCR SYBR Blue master mix with SYBR Green I fluorescent dye (Biolabmix, Novosibirsk, Russia) and IQ5 Cycler (Bio-Rad, Hercules, CA, USA). The following primers were used: MDR1_F: 5′-AGAGAATCCCCTCCAGATAAGA-3′, MDR1_R: 5′-AAGCCTATTCCATTTTGAACTTTCT-3′, GAPDH_F: 5′-GTGAAGGTCGGAGTCAAC-3′, and GAPDH_R: 5′-TGGAATTTGCCATGGGTG-3′. All measurements were performed in triplicate. Relative level of gene expression was normalized to the level of GAPDH according to the ∆∆Ct method and was determined with CFX96^TM Real-Time system (C1000 Touch^TM, Hercules, CA, USA). MDR1 mRNA expression levels in tumor cells were assessed as described previously [21 (link)].