Qpcrmix hs sybr kit

To quantify cellular mRNA in MDCK-II cells using qPCRmix-HS SYBR Kit (Evrogen, Russia), 4 μl of each cDNA preparation (1–100 ng of the above reaction), 0.4 μM of each (forward and reverse) primers, and 1× qPCRmix-HS SYBR (Evrogen, Russia) were mixed in a LightCycler 480 Multiwell Plate 96 (Roche Diagnostics, Switzerland) on ice in the dark and covered by LightCycler 480 Sealing Foil. Thermal cycling was done under the conditions as described above (Section Plasmids and primers). The quantification and data analysis was performed using the LightCycler 480 Service Software (Roche Diagnostics, Switzerland) and identified the binase action on cellular mRNA accumulation according to the relative fold-difference of expression levels.

Total RNA was isolated from formalin-fixed paraffin-embedded (FFPE) tissue sections using the RNeasy FFPE Kit (Qiagen, Valencia, CA, USA). RNA isolation from samples stored in RNAlater was carried out using the QIAGEN Rneasy Kit (Qiagen, Valencia, CA, USA), following the manufacturer’s protocol. To confirm the presence of the fusion transcripts, we performed reverse transcription PCR (RT-PCR) with oligonucleotide primers designed to specifically anneal to either side of the putative RTK fusion transcript breakpoint (Supplementary Table S3). First strand cDNA was synthesized from 0.5 to 1 µg total RNA using the MMLV RT kit (Evrogen, Moscow, Russia); cDNA was then amplified using the qPCRmix-HS SYBR kit (Evrogen, Moscow, Russia), and real-time RT-PCR was carried out using CFX96 Touch™ real-time PCR equipment (BIO-RAD, Hercules, CA, USA) in a 25 μL final volume containing 1× qPCRmix-HS SYBR, 25 pmol of primer pair (Table 1), and 1 μL of cDNA per reaction. PCR products were then assessed using melting curve analysis and by electrophoresis in 2% agarose gels. For the PCR products obtained, Sanger sequencing was carried out by Evrogen (Moscow, Russia) in both directions using the same primers as for RT-PCR amplification.

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)].

Quantification of pmoA genes was performed according to a previously described method [40 (link)] on a the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, USA) using qPCRmix-HS SYBR Kit (Evrogen, Russia). Concentration of pmoA genes was estimated using the primer system highly specific to the pmoA sequences retrieved from the examined soil samples at the previous stage. A primer system was designed using Primrose software [41 (link)]: pmoA-Cl-F (5′-GCA ATA TGG CAC GCT GAT GT-3′) and pmoA-Cl-R (5′-ATG TAT TCG GGC ATC GAG GTA-3′). qPCR thermal program was adjusted experimentally according to the standard protocol: 94 °C—20”; 62 °C—20”; 72 °C—15”; primers conc.—0.5 μM; Mg++ conc.—3 mM. One of the pmoA amplicons inserted in the pGEM^®-T vector was used to generate a standard curve. Standards, samples and negative controls were run in triplicate. The correlation coefficient for standard curve was 0.997.

Total RNA was isolated using TRI-Reagent (Sigma, St. Louis, MO, USA) according to the instructions supplied with the reagent. For the synthesis of the first cDNA strand, a reverse transcription kit (Sintol, Moscow, Russia) was used in accordance with the manufacturer’s instructions. Quantitative real-time PCR was performed using iCycler IQ5 (Bio-Rad, Alfred Nobel Drive, Hercules, CA, USA) and the reaction mixture from the qPCRmix-HS SYBR kit (Evrogen, Moscow, Russia). The UBQ5 gene (AT3G62250) was used as an internal reference [43 (link)]. The RPL2 gene (ATCG00830) was used as a control for the psbD gene. Primer sequences are presented in Table A1.

In order to validate the changes in gene expression determined by RNA-seq, we selected 5 genes of TFs for qPCR analysis. Poly(A) RNA was extracted as described above, but from other individuals than used for sequencing. RNA was isolated independently three times (5 individuals per repeat) for each of the three stages of regeneration. Sequences of PCR primers are shown in the Supplementary Table S6. RNA was reverse transcribed with poly-dT primers as described in the protocol of MMLV RT kit (Evrogen, Russia). PCR reaction was performed using a qPCRmix-HS SYBR kit (Evrogen, Russia) according to the manufacturer’s protocol for CFX96 Touch system (Bio-Rad, USA). The reactions were performed on three independent RNA samples per condition. Each sample was analyzed at least twice, making sure that the difference between technical replicates was lower than 0.5 Ct. The first regeneration stage was used as a control sample. All the expression values were normalized relative to the Ct geometric mean of Tubulin and EF1a genes. The delta-delta Ct method was used to evaluate expression. Primer efficacy was evaluated using ten serial dilutions (1:2) of cDNA combined from all samples in CFX Manager v3.1 software (Bio-Rad, USA). Specificity of the primers was determined by Sanger sequencing of target amplicon.