Sybr green qpcr assay

Total RNA from cultured cells or fresh surgical tissues was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) was performed using the All-in-One™ miRNA qRT-PCR detection kit (GeneCopoeia, Rockville, MD, USA) for miR-29c and small nuclear RNA U6, which was used as an endogenous control. The relative expressions of mRNA were detected by SYBR green qPCR assay (Bio-Rad Laboratories Inc, Hercules, CA, USA), and β-actin was used as an endogenous control. All qRT-PCR was performed on ABI 7500 thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). Primers used were as follows: β-actin, 5′-AGTGTGACGTGGACATCCGCAAAG-3′ (forward), 5′-ATCCACATCTGCTGGAAGGTGGAC-3′ (reverse); ITGB1, 5′-AATGTAACCAACCGTAGC-3′ (forward), 5′-CAGGTCCATAAGGTAGTAGA-3′ (reverse). The specific primers for miR-29c (HmiRQP0376) and U6 (HmiRQP9001) were purchased from RiboBio (Guangzhou, PRC). The relative expression levels were calculated using the 2^−ΔΔct method.

The expression levels of relevant mRNAs in HUVECs were measured by qRT-PCR assays. Concretely, total RNA was separated from HUVECs using TRIzol reagent (Invitrogen, Carlsbad, California, USA). NanoDrop-2000c spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) was used to determine the amount of RNA, and the integrity was determined by 1% agarose modified gel electrophoresis. Then, 1 μg of separated RNA was reverse transcribed into cDNA with the PrimeScript RT Master Mix Perfect Real Time (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. qRT-PCR assay was conducted using SYBR green qPCR assay (BioRad, USA) on the ABI Prism 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA) following the reaction conditions: 30 cycles of hot start at 94℃ for 1 min, denaturation at 94℃ for 1 min, annealing at 50℃ for 45 s, and elongation at 72℃ for 2 min, then final elongation at 72℃ for 8 min. The sequences of primers were showed in Table 1 and synthesized by Gene Pharma (Shanghai, China). GAPDH or U6 served as internal reference, and the data were processed by the comparative 2^−ΔΔCt method (15 (link)).

Recombinant rAAV8 viruses were generated in HEK293T cells and purified using iodixanol gradient ultracentrifugation [28 (link),29 (link)]: 1.8 × 10⁸ HEK293T cells were cultured in a ten chamber CellStack (Corning, USA) with DMEM + Glutamax (Gibco, Thermo Fisher Scientific, USA) supplemented with 10% FBS and 1% penicillin G/streptomycin. After 48 h, a 1:1:1 M ratio of pAAV-D377YmPCSK9-bGHpA plasmid [30 (link)] the rep-cap AAV8 helper plasmid; an adenoviral helper plasmid was mixed and transfected using polyethylenimine (PEI) (Polysciences Inc., Warrington, PA 18976 USA). Then, 72 h after transfection, the cells were harvested in 3 ml lysis buffer and lysed by four freeze–thaw cycles. The vector particles were purified using an iodixanol (Progen, Germany) gradient consisting of four phases with decreasing density (60%, 40%, 25%, 15%) spun at 50,000×g for 135 min at 4 °C. Approximately 3 ml of the 40% phase, in which predominantly full virus particles accumulate, were recovered with a 27G needle. Finally, the vector solution dialyzed into PBS (Zeba Spin Desalting Columns 7K MWCO, Thermo Scientific, USA) and concentrated (VivaSpin 10K MWCO, Sartorius, Germany). Viral titer was quantified as genome copy numbers per ml using a qPCR SYBR-Green assay (Biorad) with primers bGHpA-fw 5′ and bGHpA-rev (sequences in Suppl. Table 3) [31 (link)].