Sybr green polymerase chain reaction master mix

RNA was isolated with the RNeasy Mini Kit (Qiagen Sciences, Valencia, CA) and reverse transcription of 1.5 μg RNA was performed with Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Quantitative reverse-transcription polymerase chain reaction was carried out using the Applied Biosystems Prism 7900HT Sequence Detection System and Sybr Green polymerase chain reaction Master Mix (Applied Biosystems, Foster City, CA). All experiments were performed in triplicate, and the results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a reference housekeeping gene. Primer sequences for quantitative reverse-transcription polymerase chain reaction are listed in Table 5.

RNA isolation was performed with the RNeasy Mini Kit (Qiagen Sciences, Valencia, CA). After DNase treatment, reverse transcription of 1.5 μg RNA was performed with Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Quantitative reverse-transcription polymerase chain reaction was carried out using the Applied Biosystems Prism 7900HT Sequence Detection System and Sybr Green polymerase chain reaction Master Mix (Applied Biosystems). All experiments were performed in triplicate, and the results were normalized to the expression of the glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene. Primer sequences for quantitative reverse-transcription polymerase chain reaction are listed in Supplementary Table ST1.

For cell isolation from the hydrogel and scaffold, they were treated with collagenase and hyaluronidase (Sigma-Aldrich). The total RNA was extracted with a RNeasy-kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. A total of 2 g of RNA was used for cDNA synthesis using cDNA reverse transcription kits (Applied Biosystems, Warrington, UK). The primers were designed with Primer Express Software (Applied Biosystems). The assay was performed using the ABI Prism Sequence Detection System 7800 with SYBR Green Polymerase Chain Reaction Master Mix (Applied Biosystems). For analysis, the 2^−△△Ct method of relative quantification was adapted to estimate the copy numbers. The primer sequences are listed in Table 1. The human kidney-specific progenitors (CD133, CD24), representative renal progenitor (Pax2), and general physiological feature (pan-Cytokeratin) were surveyed. The epithelial cell properties were evaluated with endocytosis receptor (Megalin), ion channels (Muc-1), water channel (AQP1), and sodium-dependent glucose transport system (SGLT2). The dedifferentiation from final differentiated epithelium to the renal progenitor state and the EMT were evaluated with E-cadherin, β-catenin, and Vimentin.

BMMs treated with or without PBE (50 µg/mL) were cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 4 days. The cultured cells were washed with cold PBS, and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) on the indicated days. Reverse transcription of 1 μg of total RNA was performed using a PrimeScript™ RT reagent kit (TaKaRa Bio, Shiga, Japan) according to the manufacturer’s instructions. The ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and SYBR Green polymerase chain reaction Master Mix (Applied Biosystems) were used for qRT-PCR. For CT value analysis, the relative gene expression level was calculated using the 2^−ΔΔCt method [27 (link)]. The results were normalized to the expression of the GAPDH reference housekeeping gene. The experiments were performed in at least three independent experiments (n = 3). The primer sequences used for qRT-PCR are listed in Table 1.