MiRNA targeting gene screening technique
Cells were processed for RNA extraction, which was then isolated using
Trizol (Sigma Corporation) and chloroform. The isolated RNA was
precipitated with isopropanol. Any DNA present was eliminated by
treating RNA with DNase I (Takara Corporation). The RNA was subsequently
converted to cDNA via RT-PCR. In parallel, DH5α bacteria were cultured
in 2XYT media devoid of antibiotics, followed by their amplification and
harvesting. The bacterial cells were rendered competent using calcium
chloride, combined with glycerol, and preserved at -80°C. Plasmid
presence was confirmed through colony PCR, which encompassed preparing a
PCR reaction system, programming, and initiating the PCR. Lastly, a
quantitative PCR (qPCR) was executed, which involved setting up the
reaction system for the first strand and primers, amalgamating and
centrifuging the components, and running the qPCR.