Rna extraction kit

Total RNA was extracted by using an RNA extraction kit (Bioflux, Beijing, China) and then subjected to reverse transcription by using Primescript RT Master Mix Kit (Takara, Dalian, China) to synthesize cDNA. Real-time PCR was then performed using a Light Cycler (Roche) with a SYBR Premix Ex Taq II Kit (Takara, Dalian, China). Results were analyzed as described in the previous study (24 (link)). The primers used for qPCR was described in Table 2.

Finally, determination of the expression of inflammatory cytokines in pancreatic tissue was assessed using by RT-PCR. RNA was isolated utilizing RNA extraction kit (Bioflux, Basel, Switzerland) and RNA was transcripted into cDNA utilizing of Bioneer kit (Bioneer, Daejeon, South Korea). Amplification was conducted in a thermocycler (Mastercycler, Eppendorf, Westbury, NY). The PCR profile consisted of an initial denaturation for 5 min (94°C), followed by 40 cycles for 30 seconds (95°C) and annealing temperature for each gene (30 seconds) and the final step for 45 seconds (72°C). The PCR produces were electrophoresed on a 2% agarose gel comprising ethidium bromide. ImageJ software was employed for the densitometry analysis of the gel bands. The following primers were used: mouse MIP-2 (forward: 5-TCATAGCCACTCTCAAGGG-3, reverse: 5-TTGGTTCTTCCGTTGAGGG-3) and mouse IL-10 (forward: 5-GCGCTGTCATCGATTTCTC-3, reverse: 5-CCGTTAGCTAAGATCCCTG-3). Mouse ß actin (forward: 5-CTTGGGTATGGAATCCTGTG-3, reverse: 5-ACTGTGTTGGCATAGAGGTC-3) was used as a housekeeping gene.

Ten DEGs relevant to phenotype were selected to verify the RNA-seq data by the quantitative mRNA transcripts with real-time polymerase chain reaction (qRT-PCR) using the ABI Step One RT-PCR System (Applied Biosystems, CA) according to the manufacturer’s instructions. Total RNA samples were extracted using an RNA extraction kit (BioFlux, China). Then RNase-free DNase I was used to remove DNA contamination. Transcription of 500 ng RNA into cDNA was performed using a Revert Aid^TM First Strand cDNA synthesis kit (Fermentas, Shanghai, China) with random hexamer primers. Primers for detection of various genes are listed in Table 4. 16S rRNA gene was used as an internal control for quantification of relative gene expression. Each qRT-PCR reaction was conducted in a final volume of 50 μL. The thermal cycling profile was as follows: 94°C for 1 min, followed by 40 cycles of 94°C for 10 s, 55°C for 30 s, and 68°C for 15 s. Sterile water was used as negative control samples. The cycle threshold values (CT) were determined, and the 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)) with 16S rRNA as the reference gene was used to calculate the relative fold differences. This experiment was repeated three times.

RNA extraction kit (Bioflux, Hangzhou, China) and RT reagent Kit (Takara, Dalian, China) were respectively used to isolate total RNA and synthesize the first-strand cDNA following the corresponding instruction of kits. The qRT-PCR technique was performed as described by Wang et al. (2021) [35 (link)]. In brief, total of 20.0 μL reaction mix (10.0 μL SYBR Premix Ex Taq, 2 μL cDNA, 1.6 μL forward/reverse primers, and 6.4 μL distilled water) were amplified according to the following qRT-PCR programs: 95 °C, 15 s (step 1); 94 °C, 5 s following 60 °C, 30 s with 40 cycles (step 2); adding melting curve (step 3). A reference gene, polypyrimidine tract-binding protein (CsPTB) of tea plant [57 (link)] was used to quantify the relative expression levels of CsCAMTAs. The results were calculated by 2^−ΔCt or 2^−ΔΔCt method [58 (link)], and finally visualized as the mean values ± standard error (± SE). The qRT-PCR primers are listed in Table S2.

Plasmids and genomic DNA were extracted using the Plasmids mini kit and DNA purification kit (Tiangen, Beijing, China), respectively. PrimerSTAR HS DNA polymerase (Takara, Dalian, China) was used for PCR. Restriction endonucleases and T4 DNA ligase were purchased from Thermo Scientific (Waltham, USA). Total RNA was extracted using RNA extraction kit (Bio Flux, Beijing, China). The RNA was reversely transcribed into cDNA using the Revert Aid™ First Strand cDNA synthesis kit (Fermentas, Shanghai, China). RT-PCR was performed using the Real Master Mix kit (Tiangen, Beijing, China). The intracellular concentration of NADPH/NADP⁺ was determined by using NADPH/NADP⁺ Kit (Beyotime Biotechnology, Shanghai, China).

Expression level of mRNAs of CXCR4 and β-actin was evaluated in AGS cells. The reverse transcription-polymerase chain reaction (RT-PCR) procedures were carried out as previously reported [19 (link)]. Briefly, RNA was isolated using RNA extraction kit (Bioflux, Basel, Switzerland) and then transcribed into cDNA using Bioneer kit (Bioneer, Daejeon, South Korea). The RT-PCR was performed using primer sequences for human CXCR4 and β-actin (housekeeping gene) as listed in Table 1 and a thermocycler (Mastercycler, Eppendorf, Westbury, NY). Finally PCR products were electrophoresed on a 2% agarose gel containing ethidium bromide and gels were visualized under ultraviolet light using gel Documentation System (Bio-Rad, München, Germany).