I recently extracted RNA from developing plant leaves for the first time, as par
ID: 30654 • Letter: I
Question
I recently extracted RNA from developing plant leaves for the first time, as part of a very long and intensive experiment. The samples were extremely precious because of the amount of effort that went into obtaining them (harvesting thousands of miniscule leaves, one from each plant, to get the required mass).
I extracted the RNA with TRIzol and chloroform. Nanodrop showed excellent yield as you would expect for actively growing young tissue, but some of the samples had really low 260/230 ratios. I know this suggests phenol or salt contamination, but what can I do to clean the samples without losing any of the precious RNA? And how can I avoid the contamination in the future?
Explanation / Answer
You can clean up phenol by washing with choloroform, and then doing an isopropanol precipitation followed by a 75% EtOH wash (let me know if you'd like an exact protocol).
To avoid contamination (and sample loss), you have to be meticulous in your pipetting (which you'll get better with practice). You can always use those phase-lock tubes which basically jams a solid gel between the aqueous and organic phases so it's a lot easier to pipette.