I have extracted RNA from human muscle biopsy samples (~10-20mg starting material) using TRIzol and the chloroform/phenol method of extraction for use in RT-qPCR (SYBR green method). The samples have been resuspended in nuclease free water and quantified. The yield is good but the 260/230 ratio is quite low. I am considering re-pelleting the RNA to carry out an additional ethanol wash. Has anyone else tried this and do you have any other tips?
Thanks.
Poor 260/230 is usually guanidium/guanidine carryover from TRIzol. It's a very good thing to get rid of it, because guanidium is incredibly inhibitory to any downstream reactions you might want to do with your RNA.
For your samples as they stand, you should be able to reduce the contamination to a usable level by simply ethanol or isopropanol precipitating the RNA again. You'll lose some RNA (you almost always do) but you can include glycogen to help maximise recovery (10ug or so should do the trick).
I'd make your RNA preps up to 500ul, add 50ul of 3M sodium acetate (pH 5.5), 10ug of glycogen, and then either
500ul of ROOM TEMP isopropanol, followed by incubation for 20 mins at room temperature,
or
1500ul of ICE COLD ethanol, followed by incubation for 1hr at -80 or overnight at -20.
Then collect your precipitate by spinning at 13krpm in a chilled benchtop (4 degrees is fine) for 10-20mins, and wash twice with ice cold 70% ethanol (add 70% ethanol, spin, remove ethanol, add more 70% ethanol, spin, remove ethanol -also, add 70% ethanol gently and you won't even need to dislodge the pellet).
Then air dry 15mins and redissolve in H20. Expect around 70-80% recovery on a good day, 50-60% on a bad one (or if done without glycogen).
Note the temperatures above: isopropanol precipitations MUST be done at room temperature, because cold isopropanol is really quite good at precipitating guanidium (conversely, room temp ethanol is relatively poor at precipitating nucleotides at all, so if choosing ethanol you really do need to keep it cold).
Thus when following the TRIzol protocol, always make sure your samples and isopropanol are at room temperature when you're at the precipitation step.
Hello,
Do you make your extraction on the ice? I use to de that and use special tubes (from 5') and phase lock gel tube (for chlorophorm extraction) and barrier tips . I check the quality and quantity of my samples with a bioanalyser and Qubit.
Dear Jose Sofia, I suggested to you at first to be careful when you remove the aqueous phase after the separation. In this case is recommend do not remove completely the transparent liquid but (if you have enough material) to leave a small layer just above the "red phase". Secondly if you perform on column purification be aware when you centrifuge (you may also increase a little the time of centrifugation) but in particular when you remove the waste. Indeed during these steps there is an high risk that you carry over ethanol and other contaminats so pay attention do not spread any liquid outside the tubes.
Good luck!
Regards,
Laura
Poor 260/230 is usually guanidium/guanidine carryover from TRIzol. It's a very good thing to get rid of it, because guanidium is incredibly inhibitory to any downstream reactions you might want to do with your RNA.
For your samples as they stand, you should be able to reduce the contamination to a usable level by simply ethanol or isopropanol precipitating the RNA again. You'll lose some RNA (you almost always do) but you can include glycogen to help maximise recovery (10ug or so should do the trick).
I'd make your RNA preps up to 500ul, add 50ul of 3M sodium acetate (pH 5.5), 10ug of glycogen, and then either
500ul of ROOM TEMP isopropanol, followed by incubation for 20 mins at room temperature,
or
1500ul of ICE COLD ethanol, followed by incubation for 1hr at -80 or overnight at -20.
Then collect your precipitate by spinning at 13krpm in a chilled benchtop (4 degrees is fine) for 10-20mins, and wash twice with ice cold 70% ethanol (add 70% ethanol, spin, remove ethanol, add more 70% ethanol, spin, remove ethanol -also, add 70% ethanol gently and you won't even need to dislodge the pellet).
Then air dry 15mins and redissolve in H20. Expect around 70-80% recovery on a good day, 50-60% on a bad one (or if done without glycogen).
Note the temperatures above: isopropanol precipitations MUST be done at room temperature, because cold isopropanol is really quite good at precipitating guanidium (conversely, room temp ethanol is relatively poor at precipitating nucleotides at all, so if choosing ethanol you really do need to keep it cold).
Thus when following the TRIzol protocol, always make sure your samples and isopropanol are at room temperature when you're at the precipitation step.
Thank you for the great advice. I will follow your tips on re-purifying the RNA using a control sample and see how this works. Much appreciated! José
You could try a barrier method - Qiagen Maxtract tubes. They avoid the need for careful removal of the aqueous phase (it can be poured off). Phenol x-form protein and DNA are trapped under the wax barrier.
If using the system, I find that spinning the empty tubes first and placing them on ice prior to adding the Trisol helps get a better separation. Also you may sometimes have to increase the amt of chloroform if the wax barrier separation is not good.
http://www.qiagen.com/products/catalog/lab-essentials-and-accessories/maxtract-high-density
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