Stepone software

Reverse transcription of myocardial RNA was performed as described previously [10 (link)], with 250 ng of total RNA and 50 units of the enzyme Superscript II (ThermoFisher Scientific, Waltham, MA, USA). For relative quantification of RBM20, TTN, and RYR2 mRNA 2 µL of the reverse transcription reaction was used. As a housekeeping gene, HPRT1 was used [38 (link)]. The measurements were performed in quintuplicates at the StepOnePlus™ real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). For relative quantification, the comparative cycle threshold method (ΔΔCT) of the StepOneTM software (v2.0, ThermoFisher Scientific) was used [39 (link)]. Primer sequences and PCR conditions were previously described [10 (link)]. The RYR2 splice variant ratio corresponds to the ratio of the RYR2 splice variant with an additional 24 bp exon against the regular RYR2 splice variant. The TTN splice variant ratio corresponds to the ratio of TTN-N2B-splice variant to total TTN.

Using the diluted RNA, cDNA synthesis was performed using the QuantiTect Reverse Transcription Kit (QIAGEN). Two μl of gDNA Wipeout buffer (7x), 10 ng of template RNA and a variable volume of RNase-free water for a total of 14 μl was used in the first step. Then 1 μl of Quantiscript Reverse Transcriptase, 4 μl of Quantiscript RT Buffer (5x) and 1 μl of RT primer mix was added to the gDNA eliminated solution.
Two TaqMan reactions per time point were performed for the measure of mRNA stability post heat treatment. This allowed differentiation of either gDNA or cDNA with their corresponding probes. Real-time PCR mix included 0.5X Quantifast Multiplex PCR MasterMix (QIAGEN), 400 mM of each forward and reverse primer, 20 mM of TaqMan probe and 2.2 ng of template (gDNA or cDNA) for a final volume of 10 μl. Thermal cycling parameters used were 5 minutes at 95°C for enzyme activation, followed by 40 cycles of denaturation at 95°C for 30 seconds and 60 seconds of annealing/extensions at 60°C. The threshold was automatically set and generated with the Applied Biosystems StepOne^TM software; qPCR efficiency was also calculated automatically through the standard curves tab.

Determination of RNA levels was performed by qRT-PCR using the AB Power SYBR^® Green RNA-to-CT™ 1-Step Kit (Applied Biosystems^TM), StepOnePlus™ Real-Time PCR System (Applied Biosystems^TM), and StepOne^TM Software (v2.3, Applied Biosystems).
A mixture of 10 µl RNA (2–5 ng µl⁻¹), 10 µl SYBR^® green mix, 4.3 µl H₂O, 0.2 µl Reverse Transcriptase enzyme mix and 0.5 µl Primer mix (10 µM) was subjected to the following PCR cycler program: 30 min 48 °C; 10 min 95 °C; followed by 40× cycles of 15 s 95 °C/60 s 60 °C.
Each sample was measured in triplicate. The expression levels of all target genes were normalized to expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative expression levels were determined by calculating ΔΔC_t. Fold change was calculated as an average of ΔΔC_t of independent biological replicates (2−ΔΔC_t), while s.d. was calculated as ΔΔC_t + s and ΔΔC_t − s, where s is the pooled s.d. of the ΔC_t and ΔC_t control values ( $s=\sqrt{\mathrm{sd}{\left(\mathrm{ctt}\right)}^2+\mathrm{sd}{\left(\mathrm{ctc}\right)}^2}$ ).
The following primers were used: GAPDH—forward 5′-GGTATCGTGGAAGGACTCATGAC-3′, reverse 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′; LIF—forward 5′-CAGGAGTTGGGTCCAGATGT-3′, reverse 5′-GTCCACAATCTCCCAGAGGA-3′; Oct4—forward 5′-GATGTGGTCCGAGTGTGGTTCT-3′, reverse 5′-TTGTGCATAGTCGCTGCTTGA-3′; CtrD 16S rRNA—forward 5′-GGTATCGTGGAAGGACTCATGAC-3′, reverse 5′-TCAAATCCAGCGGGTATTAACCGCCT-3′.

For cDNA synthesis, the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific K1621) was used according to the manufacturer’s instructions. One hundred ninety nanograms of total RNA was used for cDNA synthesis. Real-time quantitative PCR (RT-qPCR) was performed on StepOnePlus Real-Time PCR system (Applied Biosystems Thermo Fisher Scientific) using PowerUp^TM SYBR^TM Green qPCR Master Mix (Applied Biosystems Thermo Fisher Scientific A25742). C_T values of each sample were determined in technical duplicates by the StepOne^TM Software (Applied Biosystems Thermo Fisher Scientific) using the fast cycle protocol. The relative expression levels of target genes were then quantified from seven biological replicates (n = 7) according to Pfaffl et al.^{71 (link),72 (link)}.
The primers for RT-qPCR are listed in Supplementary Table 1 and the following primers have been published previously:
Npas4 (mouse)^{73 (link)}Bdnf total (mouse)^{74 (link)}

2 ng of quantified cDNA prepared from three to five biological replicates were used to quantify the levels of specific RNAs such as rRNAs, snRNAs, and snoRNAs by real-time qPCR assays using target-specific primers for mature and precursor regions with standard SYBR Green Technology using Power SYBR® Green PCR Master Mix (Applied Biosystems). The qPCR assays were run in triplicate and conducted using an Applied Biosystems StepOne^TM real-time PCR system to determine the absolute quantification of individual target mRNAs. For each target, ΔΔ^-Ct method was used, in which SCR1 RNA (in the case of Random Primer) and ACT1 mRNA (in the case of oligo dT primer) were used as the internal control, and the abundance was determined from fluorescence by the StepOne^TM software version 2.2.2 (Applied Biosystems).

Relative RNA levels were determined by qRT-PCR using Power SYBR^® Green RNA-to-CT™ 1-Step Kit (Thermo Fisher, #4389986), StepOnePlus ™ Real-Time PCR System (Applied Biosystems TM), and StepOneTM Software (v2.3, Applied Biosystems). The reaction mixture contained 25 ng RNA, 12.5 μL SYBR^® green mix, 1.84 μL H2O, 0.16 μL Reverse Transcriptase enzyme mix and 0.5 μL Primer mix (10 μM) and was subjected to the following PCR cycler program: 30 min at 48 °C; 10 min at 95 °C; followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. The relative expression levels of all genes were determined by normalizing to mRNA expression of housekeeping gene Valosin Containing Protein (VCP). All samples were measured as triplicates. Following primers were used: MSH6- Forward: 5′-CCCCACCAGTTGTGACTTCT-3′, Reverse: 5′-TGTTGGGCTGTCATCAAAAA-3′; MLH1- Forward: 5′-TGAGCAGGGACATGAGGTTCTC-3′, Reverse: 5′-ACTAAGCTTGGTGGTGTTGAG-3′; VCP- Forward: 5′-AGCATTGACCCAGCTCTACG-3′, Reverse: 5′-TCTCATTGGCTACCTGTTCCAG-3′; E2F1- Forward: 5′-AGATGGTTATGGTGATCAAAGCC-3′, Reverse: 5′-ATCTGAAAGTTCTCCGAAGAGTCC-3′; RB- Forward: 5′-CTCTCGTCAGGCTTGAGTTTG-3′, Reverse: 5′-GACATCTCATCTAGGTCAACTGC-3′; TP53- Forward: 5′-CCTCCTCAGCATCTTATCCGA-3′, Reverse: 5′-TGGTACAGTCAGAGCCAACCTC-3′. The primers were purchased from Sigma-Aldrich and diluted to 10 µM. The melting temperature was determined to be 60 °C for all primers.

Genomic DNA was extracted from 100 μl peripheral blood mononuclear cells (PBMCs) using phenol-chloroform-isoamyl alcohol isolation method [23 (link)]. The quality of DNA was measured using spectrophotometer (NanoDrop 2000c, Thermo Scientific). The respective SNPs, including PNPLA3 rs738409, TM6SF2 rs58542926 and MBOAT7 rs641738, were genotyped using real-time PCR protocol based on TaqMan assays. The reaction was performed including 4 μl of 2.5X master mix (5 PRIME, Germany), 0.25 μl of 40X primers and probes mixture TaqMan SNP Genotyping Assay (assay ID:C_7241_10), Applied Biosystems, USA), 50–100 ng of genomic DNA and nuclease-free water to the final volume of 10 μl. The real-time PCR condition was performed in StepOne Plus Real-time PCR system (Applied Biosystems, USA) according to the manufacturer’s protocol. Briefly, initial denaturation was hold at 95°C for 10 min, followed by 50 cycles of amplification including denaturation at 92°C for 10 sec, and annealing/extension at 60°C for 1 min. Fluorescent signals (FAM and VIC) were acquired at the end of each cycle. Positive and negative controls were included in each experiment to confirm data interpretation. Allelic discrimination plot was analyzed using StepOne TM software (version 2.2, Applied Biosystems).