Total DNA was isolated from 200 µL medium from normal- or tachypaced HL-1 cardiomyocytes or 50 µL control patient/AF patient serum in 150 µL phosphate buffered saline (PBS) utilizing the Nucleospin Tissue kit (Macherey-Nagel, Landsmeer, The Netherlands) according to manufacturer’s instructions. Isolated DNA was used to determine DNA levels (a.u.) utilizing the CFX384 Real-time system C1000 Thermocycler (Bio-Rad, Lunteren, The Netherlands) in combination with SYBR green Supermix (Bio-Rad). Briefly, DNA, SYBR green Supermix and 10 µM forward and reverse primer-mix (Invitrogen, The Netherlands, Table 1 ) were added in a 384-well PCR plate (Bio-Rad) in triplicate per sample. Thermal cycling conditions were performed as a two-step approach using a pre-denaturating step at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min with data collection, ending with a melting curve analysis continuously from 60 °C to 95 °C. Mitochondrial DNA levels were adjusted for nuclear DNA levels (18S rRNA) [25 (link)] and analyzed using the ΔCT method.
Forward and reverse primer mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
Forward and reverse primer-mix is a laboratory reagent used in various molecular biology techniques, such as PCR (Polymerase Chain Reaction) and RT-PCR (Reverse Transcription PCR). It contains a pre-formulated mixture of forward and reverse primers that are designed to target specific DNA or RNA sequences for amplification.
Automatically generated - may contain errors
Lab products found in correlation
384 well pcr plate, by Bio-Rad (1 mentions)
Cfx384 real time system c1000 thermocycler, by Bio-Rad (1 mentions)
Sybr green supermix, by Thermo Fisher Scientific (1 mentions)
Sybr green supermix, by Bio-Rad (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time thermal cycler, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
2 protocols using forward and reverse primer mix
1
Mitochondrial DNA Quantification in Cardiomyocytes and Serum
2
Real-Time PCR Gene Expression Analysis
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The cDNA was generated from RNA using high capacity RNA-to cDNA kit (Applied Biosystems, USA) according to manufacturer's protocol. Real-time PCR reaction mixture was prepared by adding 1 μl sample cDNA (~40 ng/μl), 1 μl forward and reverse primer mix (10 μM, Invitrogen, USA), 3 μl DEPC-water and 5 μl 2X SYBR green PCR master mix (Applied Biosystem, USA) into a 96 well plate. The reaction was run by Step-One Plus real-time thermal cycler (Applied Biosystem, CA, USA) and the PCR conditions were as followed: first, a hot start at 95 • C for 15 min, then denaturation with 40 cycles of 95 • C for 15 s and finally annealing at 60 • C for 1 min. The primers used in this study (supplementary information, Table S3) were in-house designed and validated. The experiments were repeated thrice for data confirmation and reactions were measured in triplicates. The results obtained were calculated using ΔΔ C q method and recorded as fold changes.
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