Powerup sybr green master mix kit

Each qPCR assay with the samples exposed or non-exposed to fluconazole was performed in triplicate (a replicate technique to monitor the precision of real-time PCR amplification) on the StepOnePlus^TM RealTime PCR machine using the PowerUp SYBR Green Master Mix kit (Applied Biosystems). ACT1 was used as a reference gene to evaluate the transcriptional relative expression of the MDR1, CDR1, ERG11, and ERG3 genes. The specificity of the products of q-PCR reactions was evaluated through dissociation curves analysis (melting curve).
Each q-PCR quantification reaction contained 5 μΙ of 2X PowerUp SYBR Green Master Mix, 0.8 μl of each primer at a final concentration of 0,8 μΜ, 1 μl of cDNA (cDNA adjusted concentration to 1 ng/μl), the volume was made up to 10 μl with RNAse/DNAse-free water. The program consisted of an initial denaturation in two steps: 50 °C for two minutes and 95 °C for 10 minutes, followed by 40 cycles of denaturation at 95 °C for 15 seconds and an extension at 60 °C for 1 minute. To establish the cDNA dissociation temperature and identify non-specific amplification reactions, the following parameters were used: 95 °C for 15 seconds, 60 °C for 1 minute, and 95 °C for 15 seconds.

Total RNA was extracted from all of the collected organs^{13 ,17} using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and RT was performed with the PrimerScript RT Reagent Kit (TaKaRa, Japan) following the manufacturer’s instructions. Quantitative real-time RT-PCR was performed using the PowerUp SYBR Green Master Mix Kit (Applied Biosystems, USA), and samples were processed in duplicate with the following cycling protocol: 50 °C for 2 min; 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s; 60 °C for 1 min; and 95 °C for 45 s. The primer sequences used for RT-PCR targeted the envelope (E) gene of SARS-CoV-2 and are as follows: forward: 5′-TCGTTTCGGAAGAGACAGGT-3′, reverse: 5′-GCGCAGTAAGGATGGCTAGT-3′. The PCR products were verified by sequencing with the dideoxy method on an ABI 3730 DNA sequencer (Applied Biosystems, CA, USA). During the sequencing process, amplification was performed with specific primers. The sequences of the primers used for this process are available upon request. The obtained sequencing reads were linked with DNAMAN, and the results were compared with the MEGALIGN module in the DNAStar software package.

The RNA samples used for the high-throughput sequencing assays were also used for the qPCR assay. The DEMs were validated by stem-loop qRT-PCR [67 (link), 68 (link)]. Briefly, 1 μg of total RNA was reverse-transcribed to cDNA. The cDNA was synthesized with stem-loop reverse-transcribed primers for DEMs validation, using PrimeScript™ RT Master Mix (TaKaRa, Dalian, China). The cDNA was synthesized with oligo(dT) primer for DEGs validation, using ReverTra Ace® qPCR RT Kit (TOYOBO, Osaka, Japan). Next, 2 μL cDNA was used for qPCR according to PowerUp™ SYBR® Green Master Mix kit (Applied Biosystems, Foster City, CA, USA) instructions. All primers are given in Additional file 14. The small nuclear RNA (snRNA) U6 was used as endogenous internal control gene for miRNA and GAPDH was used for mRNA. ABI Step One Plus thermocycler (Applied Biosystems, CA, USA) was used to conduct qRT-PCR. Reactions were performed in triplicate, and the relative expression levels were quantified by the 2^-ΔΔCt method [69 (link)].

Total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) according to the protocol of the manufacturer. Reverse transcription was performed from 1 µg total RNA with the High Capacity RNA-to-cDNA kit (Applied Biosystems , Waltham, MS, USA). The isolated RNA would be treated with additional DNaseI (Invitrogen, Carlsbad, CA, USA), to get rid of potential DNA contamination. Quantitative real-time PCR (qRT-PCR) was carried out on ABI7900 with PowerUp SYBR Green Master Mix kit (Applied Biosystems, Waltham, MS, USA), according to the recommendations of the manufacturer. For quantification of mRNA expression levels, real-time PCR was run in triplicates for each cDNA sample, using GAPDH and 18 s RNA as the internal controls for SKMel-28 and A375 cells, respectively. For primers and annealing temperatures, see Table 1. Data were analyzed using the comparative CT (2^−ΔΔCT) method, which describes relative gene expression. For RNA-seq validation, the qPCR control was transfected with pT2-CAG-GFP instead of the KD constructs.