Absolute qpcr sybr green rox mix

High-throughput sequencing of miRNA was performed by Cloud-seq Biotechnology Co., Ltd (Shanghai, China). Isolation of miRNA from cultured cells and tissues was performed by using the Total RNA Miniprep Purification Kit (GeneMark). Grind fresh mouse kidney tissues with an electric grinder, total RNA was extracted from mRTECs, and grated kidney tissues by 1ml TRIzol and miRNA were isolated according to the manufacture’s instructions. Reverse transcription for specific miRNAs was performed using 2 μg miRNA and respective primers for reverse transcription (listed in Supplementary Table 1) according to the Stem-loop method. Quantitative real-time PCR was carried out using the ABsolute qPCR SYBR Green ROX Mix (Thermo Fisher Scientific) and the following cycling condition: 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. StepOne Plus device (Thermo Fisher Scientific) was used for detection. The snRNA U6 was used for normalization.

ChIP was performed as described previously43 (link). Briefly, fixation of cells was done with formaldehyde and DNA was subsequently sheared by sonification with a Bioruptor (high, 15 cycles: 30′′on, 30′′off) (Diagenode, Liège, Belgium). For immunoprecipitation, Dynabeads (Invitrogen) were incubated 15 min with 5 μg of specific antibodies H3K4me3 (07–473; Merck-Millipore, The Netherlands), H3K79me2 (ab3594; Abcam), H3K79me3 (ab2621; Abcam), normal rabbit IgG (ab46540; Abcam), normal mouse IgG (12–371; Merck-Millipore), anti-FLAG (f1804; Sigma-Aldrich) and anti-HA (101P-200; Covance, The Netherlands)) in 0.02% PBS–Tween-20, then unbound antibodies were washed off, and diluted sheared chromatin was added to the complex of magnetic Dynabeads-antibody (rotating overnight at 4 °C). After washing and elution with 2% SDS and 50 mmol l⁻¹ NaHCO₃, samples were treated with RNase and Proteinase K (Roche). DNA was purified using the Qiaquick DNA spin columns (Qiagen, Venlo, Netherlands) according to the protocol. Subsequently, RT–PCR was performed with AbsoluteQPCR SYBRGreenROXMix (Thermo Scientific) using specific primers (Supplementary Table 5). The % input was expressed as AE (Ct input−Ct ChIP) × Fd × 100%, where Fd is a dilution compensatory factor and AE represents the primer efficiency.

Cells were either treated or not with native LDL or Mox-LDL at a final concentration of 100 μg/mL for 24 hours. Total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science) and reverse-transcribed using Reverse Transcriptase Core Kit (Eurogentec) according to the manufacturer's instructions. For expression analysis of 94 preselected genes involved in angiogenesis, Human Angiogenesis 96-well StellARray qPCR array (Lonza Ltd., Switzerland) was used according to manufacturer's instruction with ABsolute qPCR SYBR Green ROX Mix (Thermo Scientific) on an ABI Prism 7900HT sequence detection system (Applied Biosystems). Data were analyzed with Global Pattern Recognition Data Analysis Tool (Bar Harbor Biotechnology, Trenton, ME, USA) using the internal array control housekeeping gene expression for normalization.

Total RNA was extracted following the standard Trizol RNA isolation protocol (Life technologies, Grand Island, NY) and cleaned by the RNeasy kit (Qiagen, Venlo, Netherlands). First strand cDNA was synthesized from the extracted total RNA using First Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche, Basel, Switzerland). qPCR was performed using ABsolute QPCR SYBR Green ROX Mix (Thermo Fisher Scientific Inc., Waltham, MA). Data were analyzed by the comparative CT method, and the relative mRNA expression is presented. The following individual specific primer sets were used for quantitative PCR: ase, 5ʹ-agcccgtgagcttctacgac-3ʹ and 5ʹ-gcatcgatcatgctctcgtc-3ʹ, btd, 5ʹ-gcacggacgtacgcacaccaat-3ʹ and 5ʹ-cctcggcggccaataccttct-3ʹ, dpn, 5ʹ-catcatgccgaacacaggtt-3ʹ and 5ʹ-gaagattggccggaactgag-3ʹ, elav, 5ʹ-gcggcgcgtatcccattttcatct-3ʹ and 5ʹ-tggccgcctcatcgtagttggtca-3ʹ, pntP1, 5ʹ-ggcagtacgggcagcaccac-3ʹ and 5ʹ-ctcaacgcccccaccagatt-3ʹ.

Total RNA was extracted from skin using a FastPrep tissue homogenizer (ThermoScan) and the RNeasy kit (Qiagen). Purified RNA was reverse transcribed using the SuperScript II kit (Invitrogen). Quantitative real-time PCR was performed on a 7500 RT-PCR System (Applied Biosystems) using Absolute qPCR SYBR Green ROX Mix (Thermo Scientific) with a final primer concentration of 200 nM. Primer sequences: Actb primer: 5′-TGACAGGATGCAGAAGGAGATTA-3′/5′-AGCCACCGATCCACACAGA-3′; Dkk3 primer: 5′-TCCCATTGCCACCTTTGG-3′/5′-CCAGTTCTCCAGCTTCAAGTACAC-3′; Cxcl9 primer: 5′-CTTCGAGGAACCCTAGTGATAAGG-3′/5′-CCTCGGCTGGTGCTGATG-3′; Cxcl10 primer: 5′-GACGGTCCGCTGCAACTG-3′/5′-CCCTATGGCCCTCATTCTCA-3′; Ccl2 primer: 5′-AGCAGGTGTCCCAAAGAAGCT-3′/5′-GGGTCAGCACAGACCTCTCTCT-3′; Ccl5 primer: 5′-CTGCTTGCTCTAGTCCA-3′/5′-ATGCTGATTTCTTGGGTTT-3′; Gbp2 primer: 5′-GATGAACAAGCTAGCTGGGAAGAG-3′/5′-CCTTGGTGTGAGACTGCACAGT-3′. Data were calculated relative to the housekeeping gene Actb by using the 2^−ΔΔCt method.