Dnase 1 enzyme

Cultured cells were lysed using RiboEx (Genall, South Korea) and RNA was extracted guanidinium-thiocyanate phenol-chloroform separation protocol, according to the manufacturer's instructions (Chomczynski and Sacchi, 1987 (link)). To eliminate any possible DNA contamination, RNA was treated with DNase I enzyme (Fermentas, USA). One unit of DNase I enzyme and 1 μl of buffer were added to 1 μg of the extracted RNA and incubated for 30 min at 37°C. Enzyme's inactivation was performed by adding 1 μl of 50 mM EDTA and incubation at 65°C for 10 min. cDNA was synthesized using PrimeScript first strand cDNA synthesis kit (TAKARA, Japan), according to the manufacturer's protocol. In brief, 0.5 μl of RT enzyme, 2 μl of RT buffer and 1 μl of random hexamer were mixed with 5 μl of DNase-treated RNA and incubated for 15 min at 37°C, and 5 s at 85°C for enzyme inactivation. For evaluating miRNAs expression, 1 μg of total RNA was polyadenylated using polyA polymerase (NEB), according to manufacturer protocol, and reverse transcribed into cDNA using anchored-oligo dT primers (Supplementary Table 1). The expression levels of PCSK9 and the nominated miRNAs were determined using specific primers listed in Supplementary Table 1. Real-time PCR was performed using BIOFACT™ 2X real-time PCR master mix (for SYBR Green I; BIOFACT, South Korea).

For quantitative real-time polymerase chain reaction (qPCR), RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Extracted RNA was first treated with DNase I enzyme (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s protocol to remove any contaminating traces of genomic DNA. Complementary DNA (cDNA) was then transcribed by RT-PCR using random primers and the Maxima First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s protocol. qPCR was then performed using specific primers to CUL5 (forward: 5’-GAACACAAGCACCCTCGTATT-3’, reverse: 5’-TCAACGGAGTTACATTCTCGTCT-3’; IDT, Leuven, Belgium) and actin (forward: 5’-CCAAGGCCAACCGCGAGAAGATGAC-3’, reverse: 5’-AGGGTACATGGTGGTGCCGCCAGAC-3’). CUL5 cDNA was amplified and measured using the SsoFast EvaGreen Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. Cycling conditions were 95 °C (30 s, activation), 95 °C (5 s, denaturation) and 60 °C (10 s, annealing/extension) for 40 cycles for CUL5 amplification on a Bio-Rad CFX96 Real-Time System run on a C1000 Thermal Cycler platform (Bio-Rad, Hercules, CA, USA). Actin cDNA served as reference for relative quantification.

TRIzol reagent, DNaseI enzyme, Moloney Murine Leukemia Virus reverse transcriptase, and SYBR Green positron emission tomography Master Mix were purchased from Invitrogen (Carlsbad, CA, USA). The BCA protein analysis reagent kit was provided by Pierce (Rockford, USA). Polyvinylidene fluoride was provided by Bio-Rad Laboratories (Hercules, USA). Rabbit anti-human osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were purchased from Novus Biologicals (Littleton,USA), rabbit anti-human receptor activator of nuclear factor-κB (RANK), β-Actin as well as goat anti-rabbit IgG horseradish peroxidase were provided by Cell Signaling Technology (Boston, USA). The ECL reagent kit was purchased from Amersham Pharmacia Biotech (Piscataway, USA), and all other reagents were of analytical grade.