Removing phenol contamination from total RNA - (Apr/01/2009 )

I recently used the Tri-Reagent method to extract total RNA from marine larvae. During the phase separation with this reagent, the "clear aqueous layer" had a pink tinge to it. I tried spinning longer at 4C, but to no avail. Of course, the pellets I got upon precipitation were a little pink. The 260/280 is pretty low. Any suggestions for removing this pigment/phenol contamination from samples that are total RNA resuspended in DEPC-treated water? I saw some suggestions for treatment with chloroform. Do I need to increase the volume of my samples (currently 30 ul) and with what? Do they have to be salty for precipitation? I would love some help and a protocol. These samples are precious because it take a lot of time to grow up enough larvae to get enough RNA for what we need.

Was the pinkish layer on top followed by the white layer and another clear(er) layer on the bottom? If so, you may have had too much salt in your phenol-chloroform extraction. This leads to a reversal of the layers with the organic layer on top, followed by the protein layer, followed by the nucleic acid layer on the bottom. In this case, you can draw off the top layers and use the bottom aqueous layer for your further RNA prep. Perhaps more info on your specific protocol would be helpful.

Here is your protocol using Trizol.

1 Remove Media
2 Add 1 ml Trizol/well (for 35 mm plate)
3 Shake for 5 min at RT in Plate
4 Triturate 6X, transfer to fresh tube
5 Add 200 ul Chloroform/well
6 Shake 20x
7 Incubate at RT for 2 min
8 Spin at 11,900 rpm for 10 min at 4°C
9 Remove ~ 560 ul top phase
Do Not Touch Interface or Phenol Layers!!
10 Add 500 ul Isopropanol to precipitate RNA
Shake 20x
11 Spin at 11,900 rpm for 10 min at 4°C
12 Remove Fluid
13 Wash with 1 ml of 70% EtOH (in DEPC-H2O) with vortexing
14 Spin at 7,500 xg for 5 min at 4°C
15 Remove Fluid
16 Air dry pellet for ~ 5 min at RT
17 Resuspend Pellet in 50 ul DEPC-Treated dH2O
18 Store at -80°C

marinelizard on Apr 1 2009, 09:27 PM said:

Are these marine larvae colerd? During my masters I worked on polyphenols. Some of them can interact with DNA in the major groove. I use to get RNA pellets with a reddish/pink color.

You have to add chloroform befor the first Spin!

Extraction a second time with chloroform will remove phenol. Ether also works well, although it is not widely used now (danger from flamability, peroxide explosion).

I use Qiagen's RNeasy Mini Kits to clean up my RNA and I always get good 260/280's a 260/230's

Hi!
I have a problem with RNA extraction because I always have a lot of phenols (low 260/230 ratio and I can guess it for the curve after nanodrop reading) and I was sondering how to eliminte this contamination. I see here that you suggest to remove it with chloroform: can someone tell me about the amount and the procedure?

Thank you very much in advance!
Ilaria

Usually it is to add 1 volume of chloroform and shake or vortex for 20 sec. Spin at 12,000 RCF for 10 minutes. Take off upper (aqueous) layer into a new tube without touching the interface (which will contain some phenol with extra protein etc.). Repeat as necessary. Detailed protocols are all over the web, including on this site, and in many protocol books such as Molecular cloning: a laboratory manual.

What do you mean by repeat as necessary? you mean add another chloroform after transfer the aqueous phase? Is it possible to remove phenol?

I also have same problem when extracting RNA from mouse embryos using trizol.

Yes, repeat the chloroform step as many times as you need to, to remove all the protein and phenol.