Power sybr green master mix 2x

Total RNA was extracted from cell monolayers and from frozen renal tissues using the “Trizol” reagent (Invitrogen), according to the manufacturer’s instruction. Yield and purity were checked using Nanodrop (EuroClone) and total RNA from each sample was reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed on an ABI-Prism 7500 using Power SYBR Green Master Mix 2X (Applied Biosystems). The comparative Ct method (ΔΔCt) was used to quantify gene expression and the relative quantification was calculated as 2^-ΔΔCt. The presence of non-specific amplification products was excluded by melting curves analysis. The primers are listed in Table 1.

For gene expression analysis mesothelial and endothelial cells were plated (2 × 10⁵ cells/cm²) in transwells and when a stable transepithelial resistances had been obtained the medium was removed, cells were washed with PBS and then treated for 3 h in PD or control solution and then re-filled with medium for 24 h. Total RNA was extracted using the Trizol reagent (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. Yield and purity were checked using Nanodrop (EuroClone, Pero, Italy), and total RNA from each sample was reverse-transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed on an ABI-Prism 7500 using Power SYBR Green Master Mix 2X (Applied Biosystems, Waltham, MA, USA). The comparative Ct method (DDCt) was used to quantify gene expression, and the relative quantification was calculated as 2^−DDCt. The presence of non-specific amplification products was excluded by melting curve analysis. The primers used are listed in Table 2.

TRIzol™ reagent (Thermo Fisher Scientific) was used to extract the total RNA from treated and untreated leaves and root samples according to the manufacturer’s protocol. The total RNA was quantified using a NanoDrop™ spectrophotometer ND-1000 (Thermo Fisher Scientific) and the RNA integrity was checked by agarose gel electrophoresis. The total RNA was treated with DNaseI, and column purified with the RNA Clean & Concentrator™ kit (Zymo Research, Foster City, CA, United States) to remove genomic DNA contamination. Total RNA (1 μg) was used to synthesize first-strand cDNA in a 20 μl reaction volume containing 1 μl of 50 μM oligo dT primer and 1 μl of 10 mM dNTP mix, 2 μl of 10 × RT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNaseOUT™ (40 U/μl), and 1 μl of SuperScript^® III RT (200 U/μl). The GUS gene-specific primers (Supplementary Table S2) were designed for qRT-PCR using Primer3 (v 0.4.0). Real-time PCR was conducted in a 15 μl reaction volume containing, 7.5 μl of Power SYBR Green 2X Master Mix (Applied Biosystems, Foster City, CA, United States), 1 μl of cDNA (12.5 ng), 0.375 μl of each primer (10 μM), and 5.75 μl of H₂O. The real-time PCR was carried out on a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems). The results were analyzed using a standard curve method for relative expression normalized to the soybean ubiquitin gene (GmUBI3).

TRI reagent (Zymo Research) was used to extract total RNA from six-week-old transgenic Arabidopsis and soybean plants according to the manufacturer’s protocol. Total RNA was quantified using a NanoDrop™ spectrophotometer ND-1000 (Thermo Fisher Scientific) and RNA integrity was checked by agarose gel electrophoresis. Total RNA was treated with DNaseI, and column purified with the RNA Clean & Concentrator™ kit (Zymo Research) to remove genomic DNA contamination. Total RNA (1 µg) was used to synthesize first-strand cDNA in a 20 µL reaction volume containing 1 µL of 50 µM oligo dT primer and 1 µL of 10 mM dNTP mix, 2 µL of 10×RT buffer, 4 µL of 25 mM MgCl2, 2 µL of 0.1 M DTT, 1 µL of RNaseOUT™ (40 U/µL), and 1 µL of SuperScript^® III RT (200 U/µL). The TI gene-specific primers (Supplementary Table S1) were designed for qRT-PCR using Primer3 (v 0.4.0). Real-time PCR was conducted in a 15 µL reaction volume containing, 7.5 µL of Power SYBR Green 2X Master Mix (Applied Biosystems, Foster City, CA, USA), 1 µL of cDNA (12.5 ng), 0.375 µL of each primer (10 µM), and 5.75 µL of H₂O. The real-time PCR was carried out on a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems). Expression analyses were performed using a standard curve method for relative expression normalized to the Arabidopsis actin gene (ACT) for Arabidopsis and soybean ubiquitin gene (GmUBI3) for soybean plants.