U6 rna primers

Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific). Real-time qPCR reactions were carried out in a final volume of 25 µL and run in iQ™5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The reactions were initially denatured at 95°C for 1 minute followed by 35 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The data were calculated by the 2^-ΔΔCt method. miR-4262 stem-loop primer and quantitative primers, as well as U6 RNA primers (internal reference), were designed and produced by RiboBio Company.

In the early morning, the peripheral arterial blood of the elbow artery was taken on fasting, and the blood sample was stored in a centrifuge tube containing heparin. After the sample was collected for 30 minutes, it was centrifuged at 2000 r/min at 4°C for 15 minutes. The serum was separated and stored in a refrigerator at -80°C for reserve. Real-time quantitative polymerase chain reaction was used to detect the expression level of miR-155 in serum. U6 RNA primers (internal reference primers) and miR-155 stem-loop primers were purchased from Guangzhou RiboBio Co., Ltd (Guangzhou, China). The relative quantity of gene expression was expressed by 2^-ΔΔCt.

Total RNA was isolated using TRIzol reagent (Invitrogen). Real-time quantitative PCRs (qPCRs) were carried out in a final volume of 25 μl using SYBR Premix Ex Taq (TaKaRa, Dalian, P.R. China), 0.4 mM of each primer, and 200 ng of a cDNA template. miR-101-3p stem-loop primer and quantitative primers, as well as U6 RNA primers, were designed and produced by Ribobio Company. The primers for HOTAIR were designed and produced by GenScript Company. Each individual sample was run in triplicate wells. PCR amplification cycles were performed using iQ™5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The reactions were initially denatured at 95°C for 1 min followed by 35 cycles of 95°C for 15 s, and 60°C for 60 s. The data were calculated using the 2^−ΔΔCt method. Enrichment of the RNAs was normalized to the U6 RNA.