Extractrna reagent

Tissue samples were collected in 1 h after the start of stimulation, placed in 1.5 mL tubes, and frozen in liquid nitrogen. RNA isolation was performed using an ExtractRNA reagent (Evrogen, Moscow, Russia) in accordance with the manufacturer’s recommendations. To remove traces of genomic DNA, RNA samples were treated with DNase I (Thermo Scientific, Vilnius, Lithuania). Reverse transcription was performed using the MMLV RT reagent kit (Evrogen, Moscow, Russia) and murine RNase Inhibitor (New England Biolabs, Ipswich, MA, USA) as recommended by the manufacturer. An equimolar mixture of random decaprimer (Evrogen, Moscow, Russia) and oligo(dT)15 primer (Evrogen, Moscow, Russia) was used; the concentration of each primer in the reaction was 1 μM. After reverse transcription, the reaction mixture was diluted 8-fold with deionized water.

Total RNA was extracted using Extract RNA reagent (Evrogen, Moscow, Russia) and reverse-transcribed into cDNA using Mint Reverse Transcriptase (Evrogen, Moscow, Russia). qPCR was performed to determine the gene expression levels in the PDX1-expressing and Control cells on a LightCycler480 Real-Time PCR platform (Roche Applied Science, Mannheim, Germany). QPCRmix-HSSYBR was used to determine the relative RNA expression. Primer sequences are shown in Table S1. The HPRT and DDX23 genes were used as an internal control. The PCR reaction conditions were as follows: 1 cycle at 90 °C for 5 min, 40 cycles at 95 °C for 20 s, 60 °C for 20 s and 72 °C for 35 s; and 1 cycle at 95 °C for 5 s, 55 °C for 60 s, and 97 °C for 15 s. The experiments were performed in triplicate for each sample. A relative expression ratio of PDX1 was normalized by using geometric means of the HPRT and DDX23 expression levels. Calculations were performed according to Ganger et al. [19 (link)] for the relative expression ratio.

Total RNA was isolated from tumors frozen in liquid nitrogen using ExtractRNA reagent (Evrogen, Moscow, Russia). Residual genomic DNA in the samples was removed by treatment with DNAse I (NEB) for 30 min at 37 °C. RNA was isolated and cleaned with CleanRNA Standard kit (Evrogen, Moscow, Russia). The efficiency of DNAse I treatment was controlled by Real-Time PCR. Transcription of IN genes was assessed by RT-PCR using OneTube SYBR-RT PCR kit (Evrogen, Moscow, Russia) using primers specific to IN and to murine GAPDH (Supplementary Table S3). The RT-PCR reaction was performed on BioRad PCR-machine at 45 °C for 15 min, 95 °C 1 min followed by 40× [95 °C 15 s, 62 °C 20 s] cycles.

RNA was isolated using the ExtractRNA reagent (Evrogen, Moscow, Russia) in accordance with the manufacturer’s protocol. RNA quality was evaluated by electrophoresis on a 2% agarose gel with 2.2 M formaldehyde added. RNA concentration was measured using an HS Qubit RNA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) on a Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, 1000 ng of RNA was reverse transcribed using M-MulV (SybEnzyme, Moscow, Russia) according to the manufacturer’s protocol. Quantitative RT-PCR was performed using SYBR Green I on a Bio-Rad CFX96TM real-time system (Bio-Rad, Hercules, CA, USA).
In order to find optimal amplification conditions, a temperature gradient was used. The final program included 95 °C, 3′ + ((95°, 20″ + 60 °C, 20″ + 72 °C, 30″) × 39). Fragments of genes encoding the proteins ALG67575.1, ALG68134.1, and ALG67202.2 were amplified using primers 5′-GGGGCAACAATGAATGGGGT-3′ and 5′-GACGACCATAACCACGACCG-3′ for ALG67575.1, 5′-GCTGAAAAATGGGACGCAGG-3′ and 5′-TCACCCACGTTGCCATAACC-3′ for ALG68134.1, 5′-CTTTGTGATTTGGGCGGCAA-3′ and 5′-AGATGCGACAAGTGATGCGG-3′ for ALG67202.2; all primers were designed with PrimerBLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast).

Total RNA was extracted from the isolated glomeruli samples using the “ExtractRNA Reagent” (Evrogen, Moscow, Russia) according to the manufacturer’s protocol. The samples containing 1 μg of RNA were transcribed to cDNA using the random oligodeoxynucleotide primers and the “MMLV RT kit” (Evrogen, Moscow, Russia). PCR amplification was performed using the mixture containing 10 ng of reverse-transcribed product, 0.4 μM of the forward and reverse primers, and the “qPCRmix-HS SYBR + LowROX kit” (Evrogen, Moscow, Russia). The amplified signals were detected using the “Applied Biosystems^® 7500 Real-Time PCR System” (Life Technologies, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). The following RT-PCR amplification protocol was used: (I) initial denaturation at 95 °C for 5 min; (II) three-segment amplification and quantification program consisting of 38 cycles: 95 °C for 30 s, 55–56 °C for 10 s, and 72 °C for 30 s; and (III) the ABI Melt Curve program to check for the presence of a single peak and the absence of primer-dimer formation in each reaction containing a template. The primers that were used in the work are presented in Table 2. The expression of the gene encoding 18S rRNA was used as an endogenous control. The relative expression levels of the target genes were calculated by ΔΔC_t method.

mRNA was isolated with ExtractRNA reagent (Evrogen, Moscow, Russia), treated by DNAse I (Sigma-Aldrich) and purified with CleanRNA Stanadart kit (Evrogen). cDNA was synthesized by Mint reverse transcriptase kit (Evrogen). After that, qPCR was performed with ready to use SYBRGreen HS mix (Evrogen) and primers specific to the ASIC1a, ASIC2, ASIC3, ASIC4, α-ENaC and γ-ENaC genes (Table S1) on Roche LightCycler 96 amplifier (Roche, Basel Switzerland). Data were analyzed by ∆∆Ct method on LightCycler SW software (Roche) and a gene expression was normalized to the β-ACTIN, GPDH and RPL13a housekeeping genes.

Extract RNA reagent (Evrogen, Russia) was used for total RNA extraction from adult 5 days old males. To obtain the fraction of small RNA, ~25 μg of total RNA were separated using 15% polyacrylamide gel electrophoresis in the presence of Urea (8 M) following excision of small RNA fraction corresponding to 21–29 nts. Illumina TruSeq Small RNA prep kit (Illumina, USA) was used for mall RNA libraries preparation. Sequencing was done on an Illumina HiSeq 2000 platform.