Peq gold trifast fl

Three pieces of 2 mm discs from the spotted area of the filters were cut by a sterile scissor and placed in 1.5 mL Eppendorf tubes. Separate sterile scissors and pincers were used for each sample to prevent cross-contamination. Each disc was placed in 750 µL Trifast^® FL (PEQLAB, Germany), homogenized, and incubated for 5 min at RT. The Trifast FL kit (Peq GOLD TriFast™ FL, Peqlab) was used to extract the RNA from the FTA^® paper according to the manufacturer’s instructions (PEQLAB Biotechnologie GmbH, Erlangen, Germany). RNA purity and concentration were determined by NanoDrop^® ND–1000 (Peqlab Biotechnologie GmbH, Erlangen, Germany). Reverse transcription was performed using the ImProm-II™ Reverse Transcription System (Promega, Madison, WI) with random primers according to the manufacturer’s instructions. Briefly, 0.1 µM lyophilized oligonucleotides provided with the kit were dissolved in 1 mL RNase-free water, and 1 µL of the random primer solution was mixed with 4 µL viral RNA and incubated at 70°C for 5 min. After adding Buffer 5× (56 µL), MgCl₂ (67.2 µL), dNTP (14 µL), RNAsin (7 µL), ImProm II (14 µL), and H₂O (51.8 µL), the mixture was incubated at 25°C for 5 min followed by 55°C for 60 min. The reaction was terminated by heating at 70°C for 15 min and cooling on ice.

To verify hypertrophic growth, protein/DNA and RNA/DNA ratios, cross sectional cell area, and the expression level of α-skeletal muscle actin as a marker for hypertrophy were determined. The cross-sectional cell area was analyzed using an Axiovert 100M microscope connected to a camera with the appropriate software (Axio vision Rel. 4.8; Zeiss, Göttingen, Germany). 35–40 representative cells/dish were analyzed. The peqGold TriFast Fl (Peqlab, Erlangen, Germany) was used to purify RNA, DNA, and protein from cardiomyocytes (cells from a dish containing 500,000 cells were lysed). Additionally, protein and DNA was isolated from cardiomyocytes using the MasterPure Complete kit (Epicentre, Germany) for analysis of hypertrophy dependent on KH7 concentration. The concentration (ng/μL) and quality of isolated RNA and DNA were determined using the NanoDrop 1000 (Peqlab, Erlangen, Germany), and the protein concentration (μg/μL) was determined with the BCA Protein-Assay Kit (Thermo Fisher, Schwerte, Germany