Isolation and Purification of Small RNA

Total RNA was isolated from exponentially growing HEK-293 cells using the NucleoZOL RNA Isolation Kit (Takara Bio USA, Mountain View, CA) according to the manufacturer’s instructions as described (20–22 (link)). To purify sRNA (<200 nt), we performed magnetic bead-based size selection with the commercially available Mag-Bind^® TotalPure NGS magnetic beads (Omega Bio-Tek, Inc., Norcross, GA) as described previously (23 (link)). Briefly, 5 μg of total RNA was dissolved in 20 μl RNase-free molecular biology grade ddH₂O and mixed with 20 μl Mag-Bind beads (i.e. RNA/magnetic bead vol/vol ratio of 1:1). The RNA/magnetic bead mixture was incubated at room temperature for 10 min. The mixture was subjected to a magnet, and the sRNA (<200 nt)-containing supernatant was collected, while the large transcripts (>200 nt) bound to beads and were discarded. The collected sRNA was subjected to PC8 phenol/chloroform extraction, followed by ethanol precipitation. The recovered sRNA was dissolved in 20 μl RNase-free molecular biology grade ddH₂O for reverse transcription reactions, or kept at −80°C.