Random dn 10 primer

For total RNA isolation pieces of frozen samples were homogenized in TissueLyzer (QIAGEN) for 8 min at 50 Hz in Extract RNA reagent (Evrogen). Next purification steps were performed following instructions for Extract RNA reagent. Quality and purity of RNA were validated using NanoDrop 3300 SpectroPhotometer (Thermo Fisher Scientific) and electrophoresis in 1% agarose gel. RNA samples were treated with DNAseI (Thermo Fisher Scientific). cDNA template was synthesized using Random (dN)10-primer (Evrogen) and MMLV RT kit (Evrogen). Quantitative Real-Time PCR (qRT-PCR) was performed on cDNA template using commercial TaqMan Gene Expression Assays for Nppa, Fhl1, Fhl2, Actn2, Synpo2, Myoz2, Nebl, Cmya5, Ldb3, Csrp3, Ilk, and Actb genes (Applied Biosystems). Expression of Hprt1 was evaluated by qRT-PCR using oligonucleotide gene-specific primer pair. The list of TaqMan assays and primers is provided in Table 1. mRNA relative expression was quantified applying _ΔΔC_t-method using Actb and Hprt1 as housekeeping control (Livak and Schmittgen, 2001 (link)).

Lung and cardiac samples were obtained post mortem, followed by immediate freezing in liquid nitrogen for transportation. Furter storage was at −80 °C. Frozen samples were homogenized with bead mill (TissueLyzer, QIAGEN, Hilden, Germany) during 5 min in Extract RNA reagent medium (Evrogen, Moscow, Russia). Quality and quantity of RNA were confirmed with spectrophotometry (NanoDrop 3300, Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis. Extracted mRNA was reverse transcribed to get cDNA from RNA matrices. For this goal, reverse transcription mechanism was used with Random (dN)10-primer (Evrogen) and MMLV RT kit (Evrogen). The amplification of the newly synthesized cDNA was carried out by the standard PCR procedures (QuantStudio™ 5 Real-Time PCR System, Applied Biosystems, Waltham, MA, USA) in 384-well plate in the following conditions: 95 °C during 10 min, 95 °C during 15 s and 60 °C during 1 min. $2^{-△△{\mathrm{C}}_T}$ method with normalization to reference gene (GAPDH) expression was used (Table 1).