Losing my RNA pellets ! - (Feb/27/2006 )
Hi,
I am extracting RNA from muscle and liver reptile tissue using Tri-reagent. I usually get a very low yield - probably about the best I could expect would be about 30 ug. But often I get much less than this. I think one of the problems is that I am losing some of my RNA pellet during the stage when I take off the supernatant and dry it.
I wonder if anyone has some tips about the pelleting stage after the second ethanol wash (in 75% ethanol) when the pellet is small ?
I can see I have small pellets in there, but they're colourless and they don't seem to be stuck on to the inside of the tube, they seem to be loose.
Does anyone have any ideas about how to prevent them from being sucked up? I am wary about leaving too much ethanol in there and leaving it to dry too long, because all the protocols say that it is hard to redissolve when it is over-dried, so I want to take off as much ethanol as I can.
There are some options, though they may not be appropriate. You can add some carrier molecules. Something like tRNA or sonicated DNA - not great if you want pure RNA. However, you might try adding glycogen to the precipitation mixture. This forms a matrix that helps DNA (so I'd imagine RNA) precipitate. It also gives you more visible pellets. You need to cool on dry ice for some time and then use an extended full speed centrifugation step.
Other than that, all I can suggest is just improve your pipetting technique. I use a protocol that always produces invisible pellets. Provided you orientate your tubes correctly in the centrifuge and spin for a little longer than normal, you can predict the location of the pellet. Then use an double tip (a 1 mL tip shoved into a 200 µL tip) to suck the s'nat out extremely slowly. when there's about 10 µL of EtOH left, put the tube on a heat block at 55 deg C for 1 minutes. Keep checking so you get it just as the last drop has evaporated.
hi
using glycogen is very helpful and greater han using tRNA (for quantitation at first point...)
when washing 75%etoh, you can let sit 30' at -20° before pelleting.
i've added the trizol complete manual.
Hey thanks, both of you.
I've ordered the glycogen and will see how it goes.
all the best! 
smurray, I remember reading your post a while back and I just had a thought:
Your RNA may not be pelleting and may be loose because you picked up a little bit of chloroform while removing the aqueous phase. When that happens, the chloroform is at the bottom of the tube and the RNA cannot go into that phase, so it hangs out above.
Just a thought!
Hey thanks Soluene,
I'll pay attention to that!
I've ordered the glycogen and will see how it goes.
all the best!
To get the last of the ethanol off my preps, I heat a glass pipette over a bunsen and pull it into a capillary which can then be used to suck off most of the ethanol without disturbing the pellet.
Hi!
Thanks fred for the TRI protocol posted, I am facing lots of problem when trying to isolate gDNA from rat cardiac muscle. Using the same ratio of TRI i get too much turbidity in the homogenate and the precipitate formed is not soluble in subsequent wash. I will try manipulating the protocol by adding Proteinase K.
Santo
Seems like all problems here were solved, but I figured I'd add another possibility from my own experience. Using Tri-reagent I once washed and pelleted with 25% ethanol (it was late, and I was tired). There was no pellet whatsoever, as the RNA was solubilized. In order to get around the problem I had to follow ethanol precipitation protocol adding NaCl and ehtanol. In the end, I got my pellet.