384 well pcr plate

RNA was isolated using the RNeasy mini kit (Qiagen). The QuantiTect reverse transcription kit (Qiagen) was used to generate cDNA. cDNA was loaded into quadruplicate wells of a 384-well PCR plate (Bio-Rad). Reactions were run using TaqMan fast universal PCR master mix and TaqMan qPCR gene expression probes. Results are presented as quantitation cycle (Cq) values normalised using reference gene Cq values and displayed as ΔCq or ΔΔCq expression (Livak and Schmittgen, 2001 (link)). The TaqMan assays are listed in Supplementary Table 3.

The following reaction concentrations were found to be optimal: 1× Q5 reaction buffer: 0.275 μM primer and 0.25 μM template, 50 μM non-limiting dNTPs and 1.25 μM EvaGreen (Biotum). The optimal Q5 DNA polymerase (New England Biolabs) amount was template-dependent: 20 U/ml for dATP and dTTP, and 10 U/ml for dGTP and dCTP detection. The reaction components were prepared as a 2×-master mix and 5 μl of the mix was pipetted into opaque wells of 384-well PCR-plate (Bio-Rad). Then an equal volume of sample was added. The reaction set-up was prepared on ice. For initial optimization runs, the thermal cycler (CFX384, BioRad) was programmed to heat the plate to 98°C (10s) followed by cooling to the final reaction temperature (66°C was considered optimal). The baseline fluorescence was immediately read after reaching the target temperature. Thereafter, the fluorescence (SYBR Green/FAM channel of the instrument) was recorded typically once every 5 min for 75 min. Later, the assay was found more sensitive when the baseline and the end-point fluorescence were read at a temperature above the primer annealing temperature (75°C). With this approach we found it best to limit the actual reaction time at 66°C to 55 min for dATP, 40 min for dTTP and dCTP, and 20 min for dGTP detection.

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 ΔC_T method.