Interphase problems with TRIzol RNA extraction - And a question about reprecipitating RNA (Apr/12/2006 )
I have just finished searching through just about every TRIzol topic in this forum, and I did not see anyone else having this problem so here is yet another TRIzol question:
I am using a fixed angle rotor centrifuge to separate the phases during TRIzol isolation. My problem is that the interphase clings to the sides of my tubes, leaving a smear of contamination in my aqueous phase - sometimes almost all the way to the top of it! I have just been pulling the aqueous phase being careful not to touch the side of the tube, but is that ok?? Also, by chance I just found that if I let the tubes sit at room temp for about 20 minutes (so this was after I already picked the very top of the aqueous phase and then just left the tubes), the interphase slides down to its place, leaving a clean aqueous phase above. So, I tried pulling more aqueous phase into separate tubes just to see what I get. Does anyone else have this problem? IS it a problem? Should I let the tubes sit until the phases settle into place before pulling the aqueous phase?
And while I have your attention, I have another question. Some of my samples came out quite with an A260/280 of less than 1.65. Ambion's website says to reprecipitate and rewash in 2.5 vol of EtOH or 1 vol of isopropanol, but only if the salt concentrations are adjusted. Does anyone have experience with this? How can I adjust salt concentrations? Do you prefer EtOH or isopropanol?
Thanks a lot!!
exactly the same thing is happening when I use tons of cells to isolate RNA from, in other words, the more cells the more of the white interphase stuff clinging to the wall of the tube. I am trying to get most of the aquous phase by pipeting from the middle of the tube avoiding walls an interphase below. As you cant properly see how deep you can go, I take a fixed volume first, and try a second time afterwards. If I catch precipitate then I just discard it. I took some of the precipitate once, and it showed up again later on when I was drying the pellet of precipitated RNA. The conz. measurement was messed up if I remember clearly with a ratio of 6.xxx something. I guess thats a problem....
Does not help much , I guess,
for your first question.
I think that this is kind of tiny contamination. In that case, i pipett as much as i can of aqueous phase (and in this case i don't mind if LITTLE quantities of phenolic phase comes in my pipett) and add same vol of chloroform.
But i was wondering
do you centrifuge at too high speed? That can drive the pellet to go to the side.
do you homogeneizes enough your sample after addition of chloroform?
for your second question, salt adjustment :
normally, RNA is supposed to be in quite 0mM of salts. In the case of precipitation with ethanol, some salts (generaly NaAcetate pH 5.5) is required. So salt adjustement means that you need to add salts to rna, homogeneizes and add ethanol before spinning. Without salts, RNA pellet with low efficiency (as DNA does in no salts medium).
If you don't want to use salts, use butanol (10x volume of sample is recommended). Butanol doesn't need to add salts for pelleting nucleic acids.
I would agree that you should reduce centrifugation speed to the minimum recommended in the manual. This will help with the distribution of the phase
i think it happens also when you wont mix the cells after adding trizol, specially when you have lots of them. add the trizol (at least 1ml for 10cm dish), and pipet A LOT - the solution is very sticky on the begining, but you need to make sure trizol solubilizes everything, so pipet till its clearly homogenous. then let it sit for a while.. also, as fred_33 says, mix well after adding chloroform. then the white stuff should not float in your interfase.
Thanks a lot for all the tips! TRI-king, I had a feeling you would know what was going on.
I was centrifuging at 12,000 x g, the highest recommended by the manual. I will definitely reduce it. I don't see a recommended minumum - it just says, "no more than 12,000 x g".
After chloroform addition, I just shake very vigorously for 15 seconds, and then I give each tube an additional shake before setting it down to incubate.
I will also try butanol if I need to reprecipitate the pellets. I'm pretty sure I do a decent job homogenizing the cells, but I will pay more attention to that in the future. Thanks again for the advice!