I receive this type of question many times. What is the best way to extract RNA for sequencing? And here is what I would recommend:
1. Trizol is always the best choice as a reagent. Ambion’s ToTTALLY RNA and MirVana are also great. Qiagen columns will get rid of smaller RNAs so would not recommend unless you don’t care for those small stuffs (ncRNAs).
2. We have QIASymphony and I would use this for DNA isolation, but for RNA, because the smaller RNAs will be gone after QIASymphony prep, I would use classic homogenizer to lyse the tissue/cells (fyi, QIASymphony has different protocol for isolating miRNA enriched RNA).
3. I would recommend using higher throughput (robot-type) devices to avoid cross-contamination and sample loss, and here we have Bullet Blender, the little rice-cooker type of machine you can buy at <$3,000. You mix small beads and lysis buffer to your tissue (I do this on dry ice) and let them shaken crazy within this rice-cooker for 1 min and your lysis is done! I actually warm up the tube at 37C for a few minutes after the 1st blending and then blend again for 1 min to ensure guanidine-type of lysis buffer to penetrate well within the cell: at lower temp, it doesn’t penetrate well so you will not get full recovery of your RNA! 🙁
We have Bullet Blender at our core, and any investigators are welcome to use it by contributing small $ to the consumables (beads and safe-lock tubes).
Contact me anytime for the instruction how to use our “little RNA-cooker” 🙂