I am doing an experiment where I am adding RNA to Promega's TnT Quick Coupled System (rabbit reticulocyte lysate) and then performing phenol/chloroform extraction. I started with a final concentration of ~300 ng/µl of RNA in RRL (1 µl RNA in 5µl final volume), extracted in 295 µl water + 300 µl acid phenol, ethanol precipitation in 3x volume of ethanol (900 µl), and then resuspension in 10 µl water.
I ran this on a 4% denaturing PAGE and got either extremely weak or no bands. Additionally, I should point out that I had 2 controls that were not in RRL that showed similar bands to the RRL. When I redid the experiment I added a condition where I doubled the concentration of RNA and saw a strong band in this lane and the same weak or no bands in the other lanes.
I searched the literature quite a bit and could not find much information on concentrations that should be used? Does anyone have information on what the minimum thresholds should be for concentrations of RNA in phenol chloroform extraction?
You are likely running into a lower limit on ethanol precipitation. When the overall concentration of DNA or RNA is very low, the efficiency drops exponentially since the nuclei formed are too small to precipitate. The proportion of DNA or RNA recovered in ethanol precipitation is a sigmoidal curve. At low concentrations the yield is very low until one reaches a threshold. To overcome the problem of low yields at low concentrations, a carrier is sometimes added. This fits with your observation that when you double the concentration of RNA you achieve substantially better yields.
I agree with Gerard, most likely using 1 micrometer of glycogen (20 micrograms/ml) would help.
I do not understnd, however, the reason for you using acid phenol instead of tris-saturated phenol.
The other argument is that you can re-extract the phenol with additional water.
Finally, I believe that it is something you forgot, you do not mention you add any salt to the precipitation. I would add 0.3M Na-acetate (final concentration) to the aqueous phase and then add the ethanol
Hope this may help
Jose A.
Thanks for the replies.
Sorry for leaving information out. I did add 10 µg of glycogen and 0.3 M (final concentration) sodium acetate for my ethanol precipitation. Additionally, tris can directly interfere with my construct, thus the acid phenol.
Thanks again!