RNA extraction, reverse transcription and real time PCR were performed as described in our previous study [32 (link)]. Reagents for RNA extraction (ExtractRNA reagent), for reverse transcription (MMLV RT kit) and for real time PCR (HS SYBR kit) were obtained from Evrogen. Gene expression levels were assessed using the Realtime PCR BioRad CFX-96 amplifier (BioRad) with the following running program for all investigated genes: 40 cycles of melting for 10 s at 95 °C, annealing for 15 s at 57.5 °C and synthesis for 15 s at 72 °C. Melting curves analysis was applied to control specificity of reactions. The following analysis of the obtained data was performed using the Bio-Rad CFX Manager software (BioRad) with standard 2deltaCt quantification method with the use of GAPDH expression as the reference. Primer sequences are listed in Table 1 .
Primer oligonucleotide sequences
| # | Oligonucleotide | Sequence |
|---|---|---|
| 1 | GAPDH forward | 5′-GAGGTCAATGAAGGGGTCAT-3′ |
| 2 | GAPDH reverse | 5′-AGTCAACGGATTTGGTCGTA-3′ |
| 3 | BAX forward | 5′-TGCTTCAGGGTTTCATCCA-3′ |
| 4 | BAX reverse | 5′-GGCGGCAATCATCCTCTG-3′ |
| 5 | KCNJ2 forward | 5′-TCCGAGGTCAACAGCTTCAC-3′ |
| 6 | KCNJ2 reverse | 5′-TTGGGCATTCATCCGTGACA-3′ |
| 7 | MCL1 forward | 5′-GATGATCCATGTTTTCAGCGAC-3′ |
| 8 | MCL1 reverse | 5′-CTCCACAAACCCATCCCAG-3′ |
| 9 | SLC12A forward | 5′-GAGGAGATGGACAGTAACCCC-3′ |
| 10 | SLC12A reverse | 5′-CTGGCTCAGGTTGGTGTAGTT-3′ |
| 11 | WNK4 forward | 5′-GTGAAGGCTGCGGAAGACTC-3′ |
| 12 | WNK4 reverse | 5′-CTGGGTCTCCATGTCCTCCTT-3′ |