Qrt pcr kit

Total RNA was extracted from cells and PC tissues using TRIzol (15596026, ThermoFisher, USA) and converted to cDNA using PrimeScript Reverse Transcriptase Kit (D7160M, Beyotime, Shanghai, China). According to the manufacturer’s protocol, qPCR was performed using a qRT-PCR kit (QR0100-1KT, Sigma-Aldrich, USA) in a StepOne Plus Real-time PCR System (ThermoFisher). Target gene expressions were detected by the 2^−ΔΔCT method, which were standardized with U6. Each qRT-PCR experiment was conducted in triplicate and independently repeated three times. Sanger sequencing: The amplification products of circCGNL1 in a T vector were sent to Sangon (Shanghai, China) for Sanger sequencing analysis. The primers were synthesized and designed to verify the back splice junction of circCGNL1. The primer sequences used in this study are listed in Supplementary Table 1.

In line with the instruction of TRIzol reagent (T9108, Takara, Japan), the isolation of total RNA samples was extracted in ECA-109 and KYSE450 cells. Synthesis of complementary DNA (cDNA) for miRNAs was carried out using TaqMan™ MicroRNA Reverse Transcription Kit (TaKaRa, Kusatsu, Japan), and the cDNA for mRNAs was synthesized by PrimeScript RT reagent Kit (TaKaRa, Japan). RT-qPCR reaction was achieved with qRT-PCR Kit (QR0100-1KT, Sigma-Aldrich, St. Louis, MO, USA) followed by 2^−ΔΔCt method. In relevant assays, the reference gene was β-actin while the reference gene for miRNA quantification was U6 and miR-16 (in exosome experiments). Related sequence information is included in Supplementary Table S1.

For RNA extraction, the total RNAs of the cells were isolated with TRIzol reagent. The total RNAs were transcribed as cDNA by a PrimeScript® RT reagent Kit (Thermo Fisher, Massachusetts, the USA). The reaction system including primers, cDNA, dNTPs, and Taq DNA polymerase were prepared according the instruction of the qRT-PCR kit (Sigma-Aldrich, Missouri, USA). After that, qRT-PCR was performed according to the following program: denaturation at 95°C for 3 min, followed by amplification for 40 cycles at 95°C for 12 s and at 53°C for 40s and 70°C for 30s. The relative levels of miRNA or mRNA were calculated with the 2^−(ΔΔCt) method. The sequences of the primers are given in Table 1.

TRIzol reagent (Thermo Fisher) was used to isolate total RNA from HCC cells under the indicated treatments. A PrimeScript™ RT Reagent Kit was applied for cDNA synthesis. PCR was performed with a qRT‒PCR kit (QR0100-1KT, Sigma‒Aldrich) with an ABI 7300 Real-Time PCR system. Relative RNA levels were quantified with the 2^−ΔΔCt method and normalized to GAPDH. The primer sequences are as follows:
TLR4
Forward: 5′-GGACCTGAGCTTTAATCCC-3′,
Reverse: 5′-GATTTCACACCTGGATAAATCC-3′.

After being extracted from GC cells via Trizol (abs60154, Absin, Shanghai, China), total RNAs were reversely transcribed into complementary DNAs (cDNAs) using 1st Strand cDNA Synthesis Kit (11141ES10, Takara, Japan). Then, qRT-PCR Kit (QR0100-1KT, Sigma-Aldrich, USA) was used for PCR and evaluation of the relative expression levels of different RNAs: TFAP2A-AS1, RRAGD, CDK16, ZNRF1, NISCH, SOX12, FOXq1, ZNF740, KLF15, ZNF281, IRF3, hsa-miR-876-3p, hsa-miR-4516, hsa-miR-9-5p, hsa-miR-5703, hsa-miR-3131, hsa-miR-4687-3p, hsa-miR-6762-5p, hsa-miR-4434, hsa-miR-3142, hsa-miR-3657 and hsa-miR-1245b-5p. The results were calculated based on 2^−ΔΔCt method. β-actin and GAPDH served as the internal reference for this analysis.

Total RNA was extracted in SCC9 and SCC15 cells by the application of TRIzol® reagent (Takara, Japan) in line with the protocols of supplier. Prime Script™ RT Master Mixture (Takara 11141ES10, Japan) or TaqMan® MicroRNA RT kit (4366596, Applied Biosystems™, Foster City, CA, USA) was utilized for RNA reverse transcription (RT). Then, qPCR was implemented with the qRT-PCR Kit (QR0100-1KT, Sigma-Aldrich, USA) by using StepOnePlus™ Real-time PCR Systems (Applied Biosystems™). Relative expression levels were calculated using the 2^−ΔΔCt method, with U6 small nuclear RNA (U6) as the endogenous control to normalize miRNA expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous control to normalize mRNA/circRNA expression. Related primer sequences were exhibited in Additional file 8: Table S1.

qPCR was carried out as described before [15 (link)]. The total RNAs were extracted from GES-1, AGS, HGC-27 and MKN-45 cells using TRIzol reagent (T9108, Takara, Japan). After the removal of genomic DNA, the RNAs were subjected to reverse-transcription by Hifair III 1st Strand cDNA Synthesis SuperMix (11141ES10, Takara) for qPCR. The samples were measured using a qRT-PCR kit (QR0100-1KT, Sigma-Aldrich, USA). The results were calculated on a basis of 2^-ΔΔCt. Bio-repeats were implemented in triplicate.