Power syber green pcr master mix

Total RNA from was extracted from ~20 mg of snap-frozen tissue using RNeasy Fibrous Tissue Mini Kit (Qiagen) with an on-column DNase treatment. RNA was converted to cDNA using the High Capacity RNA-to-cDNA Kit (Thermofisher). The cDNA was used to perform quantitative PCR on the QuantStudio 5 Real-Time PCR System (Thermofisher) with ~10 ng of cDNA, 250 nm of forward/reverse primers (Supplementary Table 2), and Power Syber Green PCR Master Mix (Thermofisher). Amplification was performed at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 53 °C for 15 s, 60 °C for 45 s. Primers were designed to span across 2 exons of the gene (Supplementary Table 2).

Total RNA was extracted using the TRIzol Reagent according to the manufacturer’s instruction. For miRNA analysis, total RNA was reverse transcribed with Taqman microRNA Reverse Transcription Kit and PCR was performed using the 7900 HT ABI PRISM and the TaqMan Universal PCR Master Mix II no UNG (Thermo Fisher Scientific). For mRNA quantization, RNA was reverse transcribed with the Super Script II Reverse Transcriptase and amplified with Power Syber Green PCR Master Mix (Thermo Fisher Scientific). Primer sequences will be supplied upon request. Relative quantification of transcripts was performed using the 2^−ΔΔCt method.

The reverse transcription was achieved using QuantiMir kit (System Biosciences, Mountain View, CA, USA), according to the manufacturer’s instructions, starting from 1 μg of total RNA. Quant Studio 12K Flex Real-Time PCR (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to detect the expression of miR395 with the following reaction mixture: 5 μL of Power Syber Green PCR master mix (Life Technologies, Carlsbad, CA, USA), 0.1 μL of forward primer, 0.1 μL of reverse primer, 1.4 μL of nuclease-free water, and 1 μL of each sample. The PCR program was set up with an initial denaturation at 95 °C for 10 min followed by 50 cycles at 95 °C for 15 s and 60 °C for 1 min. The primer sequence used to detect miR395 was obtained from [20 (link),21 (link),22 (link),23 ,24 (link),25 (link),26 (link),27 (link),28 (link),29 (link),30 (link),31 (link),32 (link),33 (link),34 (link),35 (link),36 (link),37 ,38 (link)]. The threshold method was used to analyze miR395 expression and the ΔCt values, calculated based on Hajizadeh et al. [39 (link)], involved the use of 18S rRNA as an internal standard.

Total RNA was isolated by RNeasy plus Mini Kit (Qiagen). First-strand cDNA was produced from 2 μg total RNA using SuperScript II First-Strand Synthesis Systems Kit (Invitrogen) using a mixture of oligo-dT and random hexamers oligonucleotides following manufacturer's instructions. The pluripotency of human iPSCs clone IV was evaluated by TaqMan gene expression assay (Life Technologies) using predesigned probes for pluripotent and undifferentiated gene markers (Supplementary Table 1) according to the supplier's instructions. RNA from commercially available human iPSCs was used as reference sample and the gene expression level was normalized to RAF1 housekeeping gene.
For gene expression analysis in renal induction experiments TaqMan Array Human Endogenous Control Plate (Life Technologies) was used in order to evaluate the best housekeeping gene and ELF1 gene was chosen to normalize the gene expression level. Real-time PCR assays were performed using Power Syber Green PCR master mix (Life Technologies). The primers employed are listed in Supplementary Table 2. The ΔΔCt technique was used to calculate cDNA content in each sample using the cDNA expression of the pluripotent state (d0) as a calibrator. The experiments were performed in triplicate and the results were expressed as mean ± SD.

The neurons were disrupted in QIAzol Lysis Reagent (1023537, Qiagen, Hiden, Germany), and total RNA was extracted and quantified using the RNeasy Lipid Tissue Mini Kit (1023539, Qiagen, Hiden, Germany). RNA was reverse transcribed into cDNA by using Transcriptor First Strand cDNA Synthesis Kit (04896866001, Roche Life Sciences, Indianapolis, IN, USA). The following primers were used: p62 forward 5′-TGTGGAACATGGAGGGAAGAG-3′, p62 reverse 5′-TGTGCCTGTGCTGGAACTTTC-3′, ywhaz (as control) forward 5′-GAAGACGGAAGGTGCTGAG-3′, ywhaz reverse 5′-GACTTTGCTTTCTGGTTGC-3′. Real-time quantitative PCR was performed in a 20 μL reaction volume (1 μg cDNA, 1 μL primer, 10 μL Power SYBER Green PCR Master Mix (Life Technologies, Warrington, UK)), and 8 μL RNA-free water on the Applied Biosystems 7500 fast real-time PCR system (Life Technologies, Foster City, CA, USA). Triplicate reactions were performed for each sample and data were analyzed using delta-delta method (ratio, 2^{−(ΔCT sample − ΔCT control)}) to compare relative mRNA levels. Real-time quantitative PCR cycling conditions were as follows: denaturation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, annealing/extension at 60 °C for 1 min.