It looks almost as if the interphase is floating up into the aqueous phase during my extraction procedure. It doesn't form a distinct layer; it is very irregularly shaped and juts out into the aqueous phase at certain points so when I extract the RNA by removing the supernatant, I feel as if I am also getting part of the interphase. Sometimes it looks like the upper aqueous layer is mostly cloudy.
Has anyone else had any similar experiences? When I do the extraction with tissue, there is always a pretty clear/opaque looking interphase.
Whenever I measure my samples (diluted in water), my 260/280 ratio is always >2.5, so I feel as if there is DNA contamination since DNA is supposed to reside in the interphase.
The most common cause of this problem is using too little TRIzol for the amount of sample. I would check that the amount of TRIzol is sufficient (according to the protocol) and maybe also try doubling the volume of TRIzol for the same amount of cells just to see if there is an improvement. Other possible causes are issues with the centrifugation step (too slow, too short, or too warm) or else allowing the samples to sit around between centrifugation and pipetting the aqueous phase off (if I process more than 4 samples, I usually take out 2-4 at a time and leave the others spinning.) I have also heard that the presence of certain chemicals in the sample can interfere with phase separation, but it has never happened to me so I can't say which substances would or would not interfere.
If you keep having this problem, you could consider using Phase-lock gel, which creates a physical barrier between the aqueous and interphase and makes it very easy to collect the aqueous phase cleanly. I found it very useful when I had to process lots of trizol samples.
Troncey Songer
With Trizol extraction, also make sure you use excess Trizol. I typically use 1 mL for 1.0x10^6 cells or for about 50mg of tissue. Let it rest at Room Temp for 2-3minutes. Add Chloroform and shake vigorously for 15sec and let sit for 2-3min at room temp. Spin at 14000 RPM for 15 minutes at 4 C. If the pH of your Trizol is correct you will get separation. If you Trizol is contaminated or the pH has changed, this will change were RNA and DNA separate in the phases. Low pH is needed to have RNA in the aqueous layer, while basic pH causes both RNA and DNA to be in the aqueous layer.
Thank you so much Laura and Shawn! I will definitely try the protocol again with more Trizol.
OH! Another point is that I accidentally left the Trizol bottle out at RT for about 24 hours. Would this have changed the pH of the reagent? I read that it is stable at room temp for a year so I assumed it was ok... Anyone have any experience with this?
Troncey Songer
Trizol is fine at room temp. That should have not caused any issues. The main way pH can change is oxidation or contamination. It has enough salt in it, so it should function as a semi buffered solution.
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