Standard dt15 oligonucleotide

Gene expression level was determined by reverse transcription and real-time PCR. Total RNA was isolated from renal tissue samples with ExtractRNA reagent (Evrogen, Russia). RNA quality was estimated electrophoretically in 2% agarose gel. RNA concentration was determined using NanoDrop 1000 c spectrophotometer (USA). Two micrograms of total RNA was used per reverse transcription reaction with MMLV reverse transcriptase and standard dT₁₅ oligonucleotide (Evrogen, Moscow, Russia). The synthesized cDNA was used for real-time PCR with 200 nm gene-specific primers (Table 1). Real-time PCR was carried out using DNA amplifier DTlite (DNA-Technology, Moscow, Russia) with qPCRmix-HS SYBR kit (Evrogen, Moscow, Russia). The PCR cycling mode was as follows: (1) «hot-start»: 95 °C, 5 min; (2) denaturation, 95 °C, 15 s; (3) primer annealing and DNA synthesis at 60 °C, 30 s. Stages (2) and (3) were repeated 40 times. The threshold cycle (Ct) value was determined using DTmaster software (DNA-technology, Moscow, Russia). The signal was normalized to that obtained for the gene of β-Actin (Actb). ∆Ct value was calculated by the formula ∆Ct = Ct (gene of interest) – Ct (Actb); ∆∆Ct was calculated ∆Ct (control) − ∆Ct (experiment). The 2^-∆∆Ct method was used to calculate differences in genes expression [48 (link)].