Dear all,
I would like to kindly ask for any recommendations on how to extract pure RNA from honeybee brains. After snap-freezing whole bees in liquid nitrogen, I have been smashing the whole head in 250 μL of Trizol with a probe and then proceeding with a homogenizer. Next, I centrifuge for 10 minutes at 12,000 g to pellet the heads and transfer the Trizol suspension to a new tube, and proceed with the standard protocol. In my case, the upper aqueous phase is always slightly pink/brown after phase separation, and both the 260/280 and 260/230 ratios are far from 2.0. How can I improve my protocol to get more pure results?
Thank you so much in advance!
I've had great luck with the Qiagen DNA/RNA extraction kit. Grind the heads in liquid nitrogen & then run through a Qiashredder column. Then proceed with the protocol.
Good luck!
I really understand that using Trizol is convenient for RNA isolation, however, there is a possibility that specific tissues (as yours) will react unusually to the presence of guanidine thiocyanate, which is present in the Trizol composition. Try the classic method with acidic phenol.
I like the Quiashredder approach as well, works pretty well. You could try a second extraction step to try and remove more of the contaminants, there will be some RNA loss but what you get should be cleaner. Or, add a higher volume of all your reagents as you may be reaching the limit of what can be removed by the current amount. I had to do that for RNA extraction from sweetgum tree leaves.
There should be some good protocols from the Drosophila community as well.
Hope this helps!
Hello Natalie El-Bittar
I am concerned about the upper aqueous phase which is always slightly pink/brown after phase separation. The upper aqueous phase is usually colorless and clear.
There are a few possibilities you need to consider.
1. If your sample contains fat micelles, they may pick up pigment from the TRIzol itself and cause a pinkish color. So, if you feel that your sample is thought to contain fat, the sample homogenate in TRIzol may be centrifuged prior to addition of chloroform. The fat will appear as a clear layer at the top of the supernatant. This should be pipetted off and discarded.
2. A pinkish aqueous phase may also be caused by over-dilution of the sample namely, the sample:TRIzol ratio > 1:10; as well as too much salt or protein in the sample. This can cause premature phase separation, which can be solved by adding a bit more TRIzol to the sample. Always maintain a ratio of 10:1 between the volume of TRIzol and the mass of the sample. Please note that if you isolate RNA from a pinkish aqueous phase, there are chances that it will be contaminated with DNA.
3. If you record a poor 260/280 ratio for RNA it may be because of the following.
a. Sample may have been homogenized in too small a volume of TRIzol.
b. Sample may not have been stored at room temperature for 5 minutes after homogenization.
c. Final RNA pellet may not have been fully dissolved. This may be the case if the RNA pellet has overdried.
d. There may be phenol contamination. This may occur if sample may have been centrifuged at room temperature instead of 4°C as phenol is more soluble in the aqueous phase at room temperature. If absorbance is seen at 270 nm (phenol), then sample can be ethanol precipitated to remove residual phenol.
e. If sample is dissolved in water, the ratio may be low due to the acidity of the water or the low ion content in water. The ratio can go up if the sample is dissolved in TE and the spectrophotometer is zeroed with TE.
Hope these suggestions help!
Best.
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