Rnazol reagent

Total RNA was isolated from frozen liver and jejunum using RNAzol Reagent (Molecular Research Center, RN190) according to the manufacturer's instructions. The concentration of the RNA was determined using an ND-1000 UV-Visible Spectrophotometer (Nanodrop). The integrity of the RNA was verified by evaluating ribosomal RNA bands (28S/18S) separated on a 1% denaturing agarose gel. Complementary DNA was synthesized from 2 μg of total RNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, 4374966). The cDNA was subjected to quantitative real-time PCR reaction using TaqMan™ Fast Advanced Master Mix (Applied Biosystem, 4444557) and TaqMan Gene Expression Assay containing a set of PCR primers and a TaqMan probe for cynomolgus monkey NPC1L1 and PAK1IP1 (Applied Biosystem; 4351372, Assay ID: NPC1L1, Mf02793772_m1; PAK1IP1, Mf02855023_m1). PCR was performed on Biorad C1000 Touch Thermal Cycler with the following conditions: 95°C for 30 s, followed by 40 cycles at 95°C for 10 s and 57°C for 25 s. The relative amount of target gene was normalized to the amount of PAK1IP1 (internal control) using the 2-ΔΔCT method.

RNA was extracted from freshly harvested mouse heart tissue with RNAzol Reagent, following the manufacturer’s protocol (Molecular Research Center, Inc. Cincinnati, OH, USA). At least 3 mice from each experimental group were utilized for RNA extraction. Libraries were constructed following an established protocol [25 (link)]. All the libraries were analyzed and quantified with a bioanalyzer and sequenced on an Illumina NEXTSeq 2000 system using a fee-based service at the Weill Cornell Genomic Core Facility. All sequence data of the 13 libraries were deposited in the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA916203.

Total RNA, including miRNAs, was isolated from EA.hy926 and HUVEC with RNAzol Reagent (Molecular Research Center Inc, Cincinnati, OH) according to manufacturer’s instructions. Isolation of miRNA from plasma was performed with NucleoSpin^® miRNA plasma kit (Machery-Nagel, Düren, Germany). Reverse transcription (RT) reaction of total RNA (400 ng) was performed using SuperScript™ III First-Strand Synthesis System (Invitrogen, Madrid, Spain). MiRNA cDNA was synthesized from total RNA purified from cells (200 ng) and from plasma (8 μL of each sample) using individual miRNA-specific RT primers and the TaqMan^® MicroRNA Reverse Transcription Kit (Life Technologies, Madrid, Spain). Each cDNA was amplified using the TaqMan^® MicroRNA assays together with TaqMan^® Universal PCR Master Mix, No AmpErase^® UNG (Life Technologies, Madrid, Spain). The 2^−ΔCt method was followed to calculate the relative abundance of miRNA and mRNA compared with endogenous control expression, U6 for cells miRNA and ACTB for mRNA. Plasma miRNA was normalized with geometric mean of miR-103-3p and miR-191 (Ct = Threshold Cycle; ΔCt = Ct sample gene-Ct endogenous control).

Total RNA was isolated from various tissues using the RNAzol reagent (Molecular Research Center, Cincinnati, OH, USA) according to a modified protocol (22 (link)), and reverse transcribed to cDNA using a High Capacity cDNA Reverse Transcription kit (Thermo Fisher). The amount and purity of the RNA was determined using a Nanodrop 2000 (Thermo Fisher). Real-time quantitative PCR (qPCR) was performed using SYBR green with a C1000 Touch Thermal Cycler (Bio-Rad). PCR efficiency calculated from the equation of efficiency (EFF) = 10(−1/slope) −1 and authentic PCR products were confirmed by analyses of melting curves, gel electrophoresis, and DNA sequencing. PCR data were analyzed using the absolute quantitation method, expressed as copies/μg total RNA, and were determined using Ct values of samples and a standard curve from serial known concentrations of plasmids containing different cDNA fragment of target genes. The full names of the genes and the primers used in this study are listed in Table 1.

Total RNA was isolated from HME-1 cells treated with different concentrations (0, 5 and 20 nM) of 1,25(OH)₂D for 24 h using RNAzol reagent (Molecular Research Center, Inc. Cincinnati, OH). The reverse transcription reaction was performed using poly-dT primer and moloney murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA) in a 25 μl reaction volume containing total RNA (2 μg), 1× PCR buffer and 2 mM MgCl₂, at 42 °C for 15 min followed by 95 °C for 5 min. The quantitative real-time RT-PCR was performed using IQ^™ SYBR Green Supermix in an iCycler (iCycler iQ Single-color real-time-PCR detection system; Bio-Rad, Hercules, CA). The primer sequences used to amplify human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) mRNA were sense 5′-CCT ACC CCC AAT GTA TCC GTT GTG-3 and anti-sense 5′-GGA GGA ATG GGA GTT GCT GTT GAA-3′; TRAIL mRNA primer sequences were sense 5′-TTC ACA GTG CTC CTG CAG TC-3′ and antisense 5′-CAG CAG GGG CTG TTC ATA CT-3′. Thermal cycling parameters were 94 °C for 4 min, followed by 35 cycles of amplifications at 94 °C for 30 s, 58 °C for 1 min, 72 °C for 2 min, and 72 °C for 10 min as the final elongation step. Relative levels of mRNA expression were normalized in all the samples analyzed on the levels of GAPDH amplification.