optimizing RNA isolation protocol - please help (Jul/22/2008 )
Hi everyone,
I'm isolating RNA from various FACS sorted mouse cell populations (monocytes, T cells, NK cells, B cells, etc). My numbers range from 20,000 cells to 2 million cells, and it's for eventual use in qRT-PCR reactions. I'm having some difficulty with the RNA isolation (using Invitrogen's TRIzol reagent and accompanying protocol). A couple of issues I'm having:
I only occasionally see a pellet after centrifuging after the ethanol wash, and haven't really seen anything during the isopropanol precipitation (this is expected for my smaller samples). But, the last pellet I did see came loose and moved around the tube when I was removing the ethanol supernatant. This makes me hyper paranoid that I'm going to discard the RNA when the pellets aren't visible. As someone pointed out elsewhere, the protocol states not to exceed speeds of 12,000 x g for the precipitation and 7500 x g to pellet out of the ethanol. Are higher speeds to get a more stable/sticky pellet acceptable / useful?
I'm getting poor ratios- 260/280 is often <1.5. I know allowing the pellet to completely dry decreases it's solubility, which would account for a low ratio, but when I can't see a pellet how can I know when it reaches the appropriate dryness?
My 260/230 ratios range from 0.59 to 0.81, is this acceptable?
I've read other threads on this forum that recommend additional chloroform steps, more ethanol washes, glycogen addition, and doing the procedure on ice. Any other tips to improve the protocol?
Any help whatsoever would be very greatly appreciated.
Sincerely,
Kelsey
I'm isolating RNA from various FACS sorted mouse cell populations (monocytes, T cells, NK cells, B cells, etc). My numbers range from 20,000 cells to 2 million cells, and it's for eventual use in qRT-PCR reactions. I'm having some difficulty with the RNA isolation (using Invitrogen's TRIzol reagent and accompanying protocol). A couple of issues I'm having:
I only occasionally see a pellet after centrifuging after the ethanol wash, and haven't really seen anything during the isopropanol precipitation (this is expected for my smaller samples). But, the last pellet I did see came loose and moved around the tube when I was removing the ethanol supernatant. This makes me hyper paranoid that I'm going to discard the RNA when the pellets aren't visible. As someone pointed out elsewhere, the protocol states not to exceed speeds of 12,000 x g for the precipitation and 7500 x g to pellet out of the ethanol. Are higher speeds to get a more stable/sticky pellet acceptable / useful?
I'm getting poor ratios- 260/280 is often <1.5. I know allowing the pellet to completely dry decreases it's solubility, which would account for a low ratio, but when I can't see a pellet how can I know when it reaches the appropriate dryness?
My 260/230 ratios range from 0.59 to 0.81, is this acceptable?
I've read other threads on this forum that recommend additional chloroform steps, more ethanol washes, glycogen addition, and doing the procedure on ice. Any other tips to improve the protocol?
Any help whatsoever would be very greatly appreciated.
Sincerely,
Kelsey
If you start with not a lot of cells, why don't you use RNeasy from QIAGEN?
I can extract RNA from about down to 0.1 million cells
I'm getting poor ratios- 260/280 is often <1.5. I know allowing the pellet to completely dry decreases it's solubility, which would account for a low ratio, but when I can't see a pellet how can I know when it reaches the appropriate dryness?
Old memories starting to catch up with me...
First, you should always know where to expect the pellet. Hence, always put the tubes in the centrifuge in the same way.
I had the same fear, that my preciuos pellets will go loose. In my hands, up to 12 000 g for 10-15 mins was just fine. You could also use glycerol, if you wish.
260/280 < 1.5 you can discard immediately.
A bit of EtOH will not hurt, so dont worry to much if you have a droplet or two.
What I used to do is I discarded the supernatant, then I did quick-spin and pipetted as much as I could off. Basically, I managed to suck up everything.
Observe your pellets carefully. Normally, they should be snow-white. When they start to become translucent on the edges, and yellowish in the middle, they are dry and you should resuspend.