Taqman fast universal pcr master mix

For RNA isolation, Trizol Reagent (ThermoFisherScientific, Waltham, MA, USA) was used according to the manufacturer’s instructions. Samples were incubated with 0.1 U/µL DNAseI (ThermoFisherScientific, Waltham, WA, USA) prior to cDNA synthesis using random hexamer primers and MLV-reverse transcriptase (Promega, Madison, WI, USA). Using TaqMan Fast Universal PCR Master Mix (ThermoFisherScientific, Waltham, MA, USA) as well as primers and probes of TaqMan gene expression assays (ThermoFisherScientific, Waltham, MA, USA), qPCR was performed on a StepOnePlus real-time PCR System ((ThermoFisherScientific, Waltham, MA, USA). CVB3 sequences for primer/probe-combinations used were 5′-CCCTGAATGCGGCTAATCC-3′ (sense), 5′-ATTGTCACCATAAGCAGCCA-3′ (anti-sense) for the primers and 5′-FAM-TGCAGCGGAACCG-MGB-3′ for the probe. The housekeeping control employed was mHPRT, with the sequences 5′-ATCATTATGCCGAGGATTTGGAA-3′ (sense), 5′-ATTGTCACCATAAGCAGCCA-3′ (anti-sense) for the primers and 5′-FAM-TGGACAGGACTGAAAGACTTGCTCGAGATG-MGB-3′ for the probe.

After siRNA treatment for 24‐48 hours or K‐563 treatment for 24 hours, total RNA was extracted from cells using an RNeasy plus kit (Qiagen), following the manufacturer's instruction. The total RNA was reverse‐transcribed using VILO reagent (Thermo Fisher Scientific). Real‐time reverse transcription polymerase chain reaction (RT‐PCR) was performed with TaqMan Gene Expression Assay (Thermo Fisher Scientific), and TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific) or TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). The transcript levels were determined using the ABI PRISM or Applied Biosystems 7500 Fast Real‐Time PCR system (Thermo Fisher Scientific). The level of each mRNA was normalized to the level of GAPDH mRNA.

RNA was extracted by first lysing cells in QIAzol (QIAGEN) and subsequently using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. cDNA was generated by reverse transcription of 1 μg RNA from each sample using MultiScribe Reverse Transcriptase (Applied Biosystems). RT-qPCR was performed using TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific) and TaqMan (Applied Biosystems) reagents. The cDNA was diluted 1:4 and 2 μL of cDNA was added to each 20 μL (for Applied Biosystems StepOne 96-well system) or 12 μL (for Applied Biosystems QuantStudio7 384-well system) RT-qPCR reaction. Each sample was run in technical duplicates or triplicates for 40 cycles of PCR, and cycle threshold (Ct) values were averaged between replicates for analysis. Relative gene expression, normalized to 18S control, was calculated as fold change in 18S-normalized gene expression, compared to baseline, using the 2^-ΔΔCt method. Baseline expression, defined as fold change = 1, was set to 3D Matrigel–cultured iAEC2 levels, or if undetected, a cycle number of 40 was assigned to allow fold change calculations. Adult human lung control RNA was extracted from a healthy donor’s distal lung explant. Primers were all TaqMan probes purchased from Applied Biosystems. Specifics of primers used are detailed in Supplemental Table 3.

Total RNA was isolated from fresh vascular tissue samples after complete homogenization in TRI Reagent® (Sigma-Aldrich, MO, USA) employing TissueRuptor (Qiagen, Hilden, Germany). Further purification was performed using RNeasy Mini kit (Qiagen), according to manufacturer’s specifications and quantified using a Thermo Scientific NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific, MA, USA). cDNA was obtained using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) for further analysis by quantitative RT-PCR. Transcripts encoding for FGF23, Klotho, runt-related transcription factor 2 (RUNX2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured by TaqMan real-time quantitative PCR (qRT-PCR) with TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific). TaqMan gene expression assays for each transcript (Hs00221003_m1 [FGF23], Hs00183100_m1 [KLOTHO], Hs00231692_m1 [RUNX2], Hs99999905_ m1 [GAPDH]) were analyzed in a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). The level of target mRNA was estimated by relative quantification using the comparative method (2^-ΔΔCt) by normalizing to GAPDH expression. mRNA levels were expressed as arbitrary units (a.u.). Quantification of each cDNA sample was tested in triplicate. A corresponding non-reverse transcriptase reaction was included as a control for DNA contamination.