Lightcycler 480 sybr green master kit

RNA was extracted from 200 µL of aliquoted serum samples using the miRNeasy Serum/Plasma RNA isolation kit (Qiagen, Hilden, Germany). Then, 25 pg/µL of RNA was used for cDNA synthesis, which was performed using the RT2 First Strand Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The LncProfiler qPCR Array Kit (System Biosciences (SBI), Palo Alto, CA, USA) was used to conduct LncRNA profiling analysis. The kit was provided with one 96-well ready-to-use qPCR plate containing predesigned PCR primer sets for specific lncRNAs. The qPCR reaction was performed on a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) instrument using a LightCycler 480 SYBR Green Master Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The 18S rRNA, RNU43, GAPDH, LAMIN A/C, and U6 genes were used as housekeeping genes. Relative quantification analysis was used to detect specific lncRNA profiling and then analyzed by Livak and Schmittgen’s 2^−ΔΔCT (delta-delta Ct) method. Relative ratio values were presented. A result of 0 to 1 was evaluated as downregulated and >1 was evaluated as upregulated [12 (link)].

Input and co-immunoprecipitated RNAs were used to make first strand cDNA using Superscript RT III (Invitrogen Life Technologies) according to manufacturer's instructions. cDNA was used for quantitative PCR using Roche LightCycler 480 SYBR Green Master kit with gene specific primers (electronic supplementary material, table S4).

Cardiac EBs 30–50 days old and human heart samples (obtained as surplus material from donor hearts during cardiac transplantations) were lysed using RNA Blue reagent (Top-Bio, Prague, Czech Republic) according to the manufacturer's instructions, and the total mRNA was isolated using the RNeasy Micro Kit (Qiagen, Hilden, Germany). RNA concentration and purity were determined using NanoDrop (NanoDrop technologies, Wilmington, DE, USA). cDNA was synthesized by Moloney Mouse Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen, Carlsbad, CA, USA) at 37°C for 1 h followed by 5 min at 85°C. For quantitative real-time PCR (qRT-PCR), LightCycler® 480 SYBR Green Master kit (Roche, Basel, Switzerland) was used according to manufacturer's instructions, and PCR was performed, with annealing temperature 60°C and 55 cycles on LightCycler LC480 Instrument (Roche). The PCR primers (Generi-Biotech, Hradec Kralove, Czech Republic) are shown in Supplementary Table 1.

gDNA was extracted with GeneJET Genomic DNA Purification Kit (Thermo Scientific) and quantitative real time PCR (qPCR) from 1.6 ng of purified gDNA was performed using Lightcycler 480 SYBR Green master kit (Roche) on a LightCycler 480 Real-Time PCR System (Roche). Primer sequences can be found in Supplementary S3 File. Target sequence primers (TFRC_B out F / TFRC_B in R, Enhancer in F / Enhancer out R for enhancer, Exon in F / Exon out R for exon) were normalised to primers GAPDH F/R amplifying a distal, non-targeted region. Another non-targeting primer set, LdhA F/R were treated in the same way. Data were normalised using the ΔΔCt method [32 (link)], incorporating primer efficiencies. The latter were estimated using a dilution series of gDNA, and efficiency calculated by the slope of the linear region only (Supplementary S4 File). We noted a decrease in efficiency at high template concentrations.
For testing the accuracy of the QC-PCR method, we used genomic templates containing known proportions of a target allele from our previous study [4 (link)]. Genomic DNA from a heterozygous clone for TFRC gene of the human, diploid cell line HCT-116, was used, combined with WT gDNA in controlled proportions.