Thermo scientific pierce magnetic rna protein pull down kit

We performed RNA-pulldown assay to detect the RNA–protein interaction using Thermo Scientific Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, USA) according to the paper and previous research [60 ].

RIP assays were carried out using the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) according to protocols. The RIP method was similar to that used for me-RIP. DGCR8 antibody (Abcam, CA, USA) was used for RIP, and qRT-PCR was performed to detect coprecipitated RNA.
For the RNA pull-down assay, concisely, miR-320a-3p synthesized by RiboBio (Guangzhou, China) and negative control RNA were labeled by using the Pierce RNA 3′-end desthiobiotinylation kit (20163, Thermo Fisher Scientific). The pull-down assay was then carried out using the Thermo Scientific Pierce magnetic RNA–protein pull-down kit (20164, Thermo Fisher Scientific) with its protocol. Briefly, the streptavidin magnetic beads were prewashed twice with 2 × volume of 0.1 M NaOH, 50 mM (nuclease-free) NaCl, and once in 100 mM NaCl. Cell lysates were prepared using standard lysis buffer and incubated with beads with rotation at 4 °C overnight to pull down the RNA complexes. Finally, the RNA-binding complexes were washed and eluted for further qRT-PCR analysis.