Rneasy mini kit

Total RNA was extracted from DCs using an RNeasy Mini Kit (Roche, Basel, Switzerland) following the manufacturer’s instructions. Reverse transcription reactions were performed using 1 μg of total RNA. Amplification was performed on a GeneAmp PCR system (PTC0200; Bio-Rad Laboratories, Hercules, CA, USA). Qauntitative polymerase chain reaction (qPCR) reactions were then run in duplicate, with each reaction containing 5 μL 2× FastStart Universal SYBR Green Master mix, 0.2 μL each target-specific primer, 0.2 μL Rox reference dye, 1 μL cDNA template, and 3.4 μL PCR-grade water in a total 10 μL reaction volume (Table). Cycling conditions were as follows: pre-denaturation at 95°C for 30 seconds, followed by 44 cycles consists of denaturation at 95°C for 5 seconds and annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, then 72°C for 7 minutes, and 4°C forever.
The extracted DNA was assayed with 1.2% agarose gels (High Strength Analytical Grade Agarose; Bio-Rad Laboratories) in TBE buffer (50 nM Tris, 50 nM borate, 1.25 nM EDTA, pH 8.2), stained with ethidium bromide for 30 minutes at 60 mA and photographed under UV light. A 100 bp to 500 bp DNA ladders were used as a molecular size marker.

Biotin-labelled miR-499a-5p or antisense RNA was transcribed using Biotin RNA Labelling Mix and T7/SP6 RNA polymerase (Roche Diagnostics, USA) and then incubated with RNase-free DNase I (Roche). After purification using the RNeasy Mini Kit (Roche), biotin-labelled RNAs were mixed with extracted Caki-1 and 786-O cell nuclear proteins and subsequently subjected to incubation with streptavidin agarose beads. The enrichment of lncRNA GIHCG was analyzed using qRT–PCR.

Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase were used for in vitro transcription of biotin-labeled MALAT-1, the products of which were purified with RNeasy Mini Kit (Roche). Radio-immunoprecipitation assay buffer was utilized to prepare the cell lysate and then the cell lysate was incubated with the biotinylated RNA biotin-labeled RNAs at 4°C for 4 hours. The washed streptavidin agarose beads were added into each binding reaction to incubate for 1 hour at room temperature. Beads were washed five times and the retrieved proteins were run on SDS-PAGE gels for Western blot.