Abi viia7 qpcr system

The RNA extraction was performed as previously described [38 (link)]. The sequences of primer pairs used in this assay are shown in Additional file 1: Table S2. Q-PCR was conducted by amplifying 20 μl of diluted cDNA with the SYBR Green Q-PCR kit (Vazyme no.Q711–02, Nanjing, China) on an ABI ViiA 7 Q-PCR System (Applied Biosystems, Waltham, MA). Each sample was run in triplicate and PCR reactions without the addition of the template were used as blank controls. The relative quantification of the expression of the target genes was measured using glyseraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control.

All RNA from tissues and cells were extracted with the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol with modifcation. cDNAs were synthesized using Rever Ace qPCR RT Kit (TOYOBO). Real-time PCR was performed using SYBR Green Realtime PCR Master Mix (Roche) and the ABI ViiA7 qPCR System (Applied Biosystems). Chromatin immunoprecipitation (ChIP) was performed to investigate whether EZH2 and H3K27 binding to p57 promoter. ChIP assays were performed as described previously [26 (link)]. RNA binding protein immunoprecipitation (RIP) experiments were performed using Magna RIP Kit (Millipore, Catalog No.17-701) according to the manufacturer’s instructions and a previously published RIP-Chip protocol [27 (link)]. RIP assays were carried out as described previously [26 (link)]. All primers were in Supplementary Table 1.

Total RNA was extracted from circulating leukocytes using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using PrimeScript RT Master Mix (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Quantitative PCR was performed using SYBR Green Realtime PCR Master Mix (Roche, Mannheim, Germany) and the ABI ViiA7 QPCR System (Applied Biosystems, Carlsbad, CA, United States). Specific primers used for the reaction are as follows: AC131056.3 -001-F, 5′-aacagatagcccagggcatttt-3′, AC131056.3-001-R, 5′-ccca cgtcctcctcattcaca-3′; HOTAIRM1-F, 5′-gatttggagtgctggagcgaaga-3′, HOTAIRM1-R, 5′-gggttcaggcaaaacagacctc-3′; lnc-MOK-6:1-F, 5′-gtcaatttttctttcttctcttgc-3′, lnc-MOK-6:1-R, 5′-ctcctttattcttcgtt cctccaa-3′; RF01976.1-201-F, 5′-cttaatgctttcggacgggg-3′, RF01976. 1-201-R, 5′-gcgattcgtcctacgctcat-3′; DEFA4-F, 5′-tccaggcaagagg tcatgag-3′, DEFA4-R, 5′-cacaccaccaatgaggcagtt-3′; NR4A3-F, 5′-tgcatgactcaatcagatttgga-3′, NR4A3-R, 5′-agcttggtgtagtcggggt tc-3′; GAPDH-F, 5′-ccagcaagagcacaagaggaa-3′, GAPDH-R, 5′-ggttgagcacagggtactttatt-3′. The relative lncRNA and mRNA expression levels were assessed using the 2^–ΔΔCt method.

Total RNA of ccRCC tissues and cell lines was extracted with the TRizol reagent (Thermo; Massachusetts, USA) according to the manufacturer's protocol. 1μg enriched tissue or cell RNA was used to synthesize cDNA through reverse transcription, which was accomplished by the RevertAid First-Strand cDNA Synthesis Kit (Thermo; Massachustts, USA). The Hieff^TMqPCR SYBR^RGreen Master Mix (Thermo; Massachusetts, USA) was used for qPCR analysis by ABI ViiA7 qPCR System (Applied Biosystems; Foster, CA). Gene primers was purchased from RiboBio (Guangzhou, China), they were listed as follows: PPT2, left primer 5'-ACCATCCCAATGCCACAGTA-3'; right primer 5'-CAACCCAAAAGAATCCCGCA-3'. GAPDH, left primer 5'-CCAGAACAGCATCCCTGCCT-3'; right primer 5'-CCTGCTTCACCACCTTCTTG-3'.

Total RNA was isolated using TRIzol extraction reagent (Vazyme, R401) and was then reverse‐transcribed using a Hiscript II Reverse Transcriptase master mixing kit (Vazyme, R201) according to the manufacturer's instructions. All primers (Supplementary Table S4) were synthesised by Tsingke Biological Technology. PCR amplicons were quantified by SYBR Green (Vazyme, Q711) using an ABI ViiA 7 Q‐PCR System (Applied Biosystems). The relative abundance was analysed using the 2 –(ΔΔCt) method and normalised against 18S rRNA.