EtOH precipitation of RNA problems - (Jul/25/2006 )
I'm trying to concentrate my total RNA prep with sodium acetate/ethanol precipitation. This is the first time I've had to do this. Luckily I did a test run of the protocol with unimportant RNA since rather than concentrating my RNA, my final concentration was lower than what I started with! So any advice would be very welcome. Here are some points which I found:
- I use linear acrylamide (2ul) as a carrier/co-precipitant
- use a 1/10 volume of 3M Na acetate and 4 volumes of 100% EtOH
- Incubate overnight at -20oC
- Centrifuge at top speed for 30 mins
- Wash pellet with 80% EtOH
Finally, I air-dry the invisible pellet and resuspend in Tris-Hcl 10mM pH7.5 and quantify RNA concentration with a 1 in 200 dilution.
Has anyone had similar experiences or have any suggestions as to what I can do?
I mainly followed Bartel’s protocol (http://web.wi.mit.edu/bartel/pub/protocols/miRNACloningUpdate0705.pdf)
For RNA precipitation, using 0.3M NaCl + 2~2.5 times EtOH (>2 hours).
You might also added glycogen 1ug/mL for precipitate small amount of RNA
I suggest a few changes:
use linear acrylamide (2 ul) as a carrier/co-precipitant.
use a 1/10 volume of 3 M Na acetate (or 1/3 volume of 7.5 M ammonium acetate) and 2.5 volumes of 100% EtOH.
Incubate overnight at -20oC (dry ice/ethanol bath for 20 minutes is almost as good).
Centrifuge at top speed for 30 mins.
Wash PELLET with 70% EtOH (if using a microfuge tube, the rinse volume should be 1 mL).
Do not try to reuspend the pellet before performing the 70% ethanol rinse. Invert a few times and chill in dry ice/ethanol bath for 10 minutes. Spin for 5 minutes. Carefully remove all liquid, leaving behind the pellet. Air dry or SpeedVac (but do not overdry). Resuspend the pellet in 10 mM Tris, pH 7.5.
i prefer to use glycogen as a carrier. 1µl of 50mg/ml stock solution for 1ml of RNA preparation.
You should also test with a sample of "junk RNA" to use butanol.
Starting with an eppi and 100µl RNA :
add 1ml buOH. Vortex well.
Complete wih 400µl buOH. Homogenize by inverting or if possible vortex.
Spin 2000g 1'.
Normally 2 phase should appear. Upper is buOH and should be discared with other solvents.
Lower one is aqueous, and should be <<100µl.
Repeat the upper procedure.
The RNA pellet should appear after this step (eventually a 3rd one may be necessary.
Wash with 70% EtOH at least 1ml (spin is done at top speed for 5')
BuOH is far more hydrophobic and doesn't need any carrier.
Try to test this method.
(it's also the one i use in case of a good 260/280 ratio and a non-that-good 260/230 ratio)
Hi miRNA man,
As tfitzwater says, don't resuspend your pellet after the first spin. This is where you are losing your RNA. When you do this, then add only ethanol, your RNA will not reprecipitate out of solution. You need the salts for this. Your protocol may be OK as it is, just wash your first pellet in 70-80% ethanol (incubate at low temp if you like, but may not be necessary) and respin. Then resuspend the pellet. The 70-80% ethanol is for removing the salts from the initial precipitation step, your pellet is not supposed to resuspend.
Thanks to everyone for your replies and tips. I'll try out your suggestions, including using the BuOH Fred suggested.
My current protocol (from Ambion) said that I should "repeat the 80% ethanol wash if needed to remove the large salt pellet from the sample" and therefore thought that the pellet had to disappear. Well I'll try out your suggestions, hopefully with better results this time! One further question - do you think that the salt pellet would make the tris reconstitution buffer to concentrated? I'm going to use the RNA samples for microarray.