Rnase free and yeast trna

The cells were transfected with biotinylated WT miR‐23a‐3p and MUT miR‐23a‐3p (50 nmol L⁻¹ each). Forty‐eight hours later, the cells were isolated and rinsed with PBS, followed by incubation with the specific lysis buffer (Ambion, Austin, TX, USA) for 10 minutes. The lysate was incubated with the M‐280 streptavidin magnetic beads (Sigma‐Aldrich, St. Louis, MO, USA) pre‐coated with RNase‐free and yeast tRNA (Sigma‐Aldrich, St. Louis, MO, USA) at 4°C for 3 hours, and then rinsed twice with cold lysis buffer. RT‐qPCR was conducted to determine the expression patterns of LINC00472 and FOXO3.

Biotin-labeled miR-27b-3p WT and mutant MUT plasmid (50 nM each) were subjected to transfection with BGC-823 and AGS cells, respectively. After 48-h transfection, cells were subjected to 10-min incubation with specific cell lysate (Ambion, Austin, USA), Then, 50 mL sample cell lysate was subpackaged. The residual lysate was incubated with M-280 streptavidin magnetic beads (Sigma, MO, USA) pre-coated with RNase-free and yeast tRNA (Sigma) at 4 °C for 3 h, then washed twice washing with cold lysate, 3 times with low salt buffer, and once with high salt buffer. The bound RNA were purified using TRIzol reagent (Invitrogen) for further qRT-PCR analysis.

Cavernous smooth muscle cells were transfected with wide‐type (WT) biotinylated miR‐205 (50 nM, Bio‐miR‐205‐WT) and mutant (MUT) biotinylated miR‐205 (50 nM, Bio‐miR‐205‐MUT). After transfection for 48 hours, the cells were collected, rinsed with PBS, incubated for 10 minutes with specific cell lysates (Ambion, Austin, Texas, USA) and sub‐packed at a volume of 50 mL. Next, the remnant was cultured at 4°C for 3 hours with streptavidin‐biotin and magnetic beads (M‐280) pre‐coated with RNase‐free and yeast tRNA (Sigma‐Aldrich Chemical Company, St Louis, MO, USA), rinsed two times with cold lysate, rinsed three times with low‐salt buffer and rinsed once with high‐salt buffer. The miR‐205 antagonist probe served as the negative control. Total RNA content was extracted using Trizol, and the expression of AR was determined by qRT‐PCR.

RC cells were transfected with wild-type (WT) biotinylated lncRNA IRAIN (50 nM) and mutant-type (MUT) biotinylated lncRNA IRAIN (50 nM). Cells were harvested and vortexed after 48 h of incubation. Specific cell lysis buffer (Ambion, Austin, Texas, USA) was added to the cells followed by 10 min of incubation on ice. The 3-h lysate incubation was performed with M-280 streptavidin magnetic beads (Sigma) pre-coated with RNase-free and yeast tRNA (Sigma) at 4°C. Cells were washed twice with cold lysis buffer, 3 times with low salt buffer, and once with high salt buffer. Total protein was extracted with a high-efficiency RIPA buffer. Dnmt1, Dnmt3a, and Dnmt3b expression was analyzed by Western blot analysis.

Liver-cancer-resistant strain cells were transfected with transcription-labeled biotin RNA probes (50 nM each). Cells were collected, washed with PBS, and vortexed after being left to transfect for 48 h. Cells were later incubated with a specific cell lysis buffer (Ambion, Austin, TX, USA) for 10 min. The lysate was incubated with M-280 streptavidin magnetic beads (Sigma) and pre-coated with RNase-free and yeast tRNA (Sigma) at 4°C for 3 h. qRT-PCR was used to determine whether miR-106a-5p and miR-372-5p were enriched in the FER1L4 group.

To verify the binding relationship between miR-143 and MALAT1, RNA-pull down assay was implemented. Three biotin-labeled miRNA sequences Bio-miR-143-WT, Bio-miR-143-MUT, and Bio-miR-NC were designed and entrusted to GenePharma Company (Shanghai, China). These biotinylated oligonucleotides were transfected into cells for 48 h. Then, the cells were harvested and incubated with a specific cell lysate (Ambion, Austin, Texas, USA) for 10 min. After that, the lysate was hatched with M-280 streptavidin beads pre-coated with RNase-free and yeast tRNA (all from Sigma) at 4 °C for 3 h, then cleaned twice with a cold lysis solution, three times with a low salt buffer, and once with high-salt buffer solution. An antagonistic miR-143 probe was established as a NC. Total RNA was abstracted with Trizol and then MALAT1 enrichment level was tested using RT-qPCR.