I have already tried different methods to extract RNA from yeast polysomal fractions, like phenol extraction-LiCl precipitation, or total RNA extraction kits (silica columns). In all cases I find that the yield is very low in those fractions where there is little RNA to begin with.
I normalize with spike-in RNAs, but then the recovery of the spike-ins is very small in some of the fractions. Therefore, I would like to know if there is any method or kit that works better than others to increase the yield when working with low levels of RNA, like in polysomal fractions. Thanks in advance
We typically wash polysome fractionator through with DEPC water and use DEPC water in the sucrose gradients (and throughout). We use Trizol to precipitate the sucrose fractions. This usually works well.
We were worried about variations in rRNA/mRNA ratios across the gradient influencing the RNA precipitation efficiencies, but our expts with spike-ins indicated this was not a problem. I think that I have read somewhere that Trizol has a bias with very short RNAs eg miRNAs, but it did not appear to be an issue for us.
It is possible to scale up gradients if you cant load enough material on a standard 12 ml (SW41) gradient. We have used larger SW28 tubes and centrifuged at 16,900 rpm for 13 hrs. This enables loading more material.
Thank you so much for both your answers. I can check both Trizol and the Norgen Biotek kits, to see if I can increase the yield.
In our case, I don't think there is a problem with RNA degradation, since we check the RNAs after each extraction and they look good. We don't use DEPC, but all the material and solutions are prepared and used in RNase free conditions.
It is more a question of the percentage of RNA that we recover, after checking RNA concentration in each fraction before and after the extraction. It seems that both the precipitation or the silica column methods we have tried have very low efficiency when there is low initial RNA. After normalization with spike-ins you correct these variations, and the qPCR profiles look OK, but I just wondered if other methods were more efficient, to begin with.
Also, it is true that loading more RNA in bigger tubes could be a solution.
Thanks again for your answers.
Dear all, did someone already use a urine cell-free Norgen Biotek kit for RNA extraction from polysome fraction? It seems a good option for 500ul fractions but I would like to have your opinion about it. Thank you
I know this is really late, but if you run it through a density gradient fractionator, you will get mostly degraded mRNA due to RNases on the tubing. I usually run two gradients, one through the machine and the other to directly extract RNA from.
Thank you very much for your answer! In our case, I don't think it is a question of degradation, as the RNA, after the extraction looks OK on a gel. We wash all the fractionator system, before and after its use, with RNase free water, and we use RNase-free water and material to prepare the gradients.
The problem we have is with the yield. We measure the RNA quantity in each fraction before and after the extraction, and the yield is in general very low, but even lower in the fractions that had less RNA in the beginning. In the end we normalize with the spike-in RNAs, and the results are coherent, but I wanted to know if other methods gave a better yield.
Anyhow, I am curious: if you don't run it through the fractionator, how do you then obtain the fractions from the other tube?
Thanks a lot again
About Maxime's question: We finally didn't try the Norgen Biotek kit. We have been using other columns, but in general with the polysome fractions, as I commented, the yield is low. Anyhow, we obtain enough RNA to detect by RT-qPCR - at least for the genes we are studying - and if you add spike-in RNA to each fraction, in order to normalize, it should be OK, even if the yield is low.
Could be our machine was not as well cleaned as yours. We ran the samples at the same time and took equal fractions from the top of the tube to extract and could compare it to the polysome and verify it with 18S and 25S rRNA probes. Doesn't match up to the fractions per se, but can be mapped to the polysomes. With low yields, we used glycogen.
@Maria Teresa, we also use rnase free conditions throughout (I don't use depc water but I know others who do), include rnase inhibitor in our gradients and lysate, and use trizol for RNA isolation from each fraction. I will admit that I have never used spike in RNAs, however there are possibly some publications that possibly you could compare your #'s to (or refer to Graham's answer above since his lab has experience with that). Recently we have added extra chloroform washes and an NaOAc precipitation step to our isolation protocol, which helped improve yield almost tenfold (I was shocked too). I've found that extra ethanol washes at the end also helps with quality. One other comment on the isolation: be sure you isolate RNA from the same volume of each fraction (not the whole fraction as there will be a drop or two variability), and be sure to extract the same volume of aqueous phase.
Finally, increasing input always helps boost yield, as expected, however, we get more than enough for most mRNA isoforms using just 2 ml gradients, so if you are using 10 ml, I can't image you would have an issue with amplification. Anyway, I'm sure this is late beyond what can be helpful- but alas the internet is a treasure trove.
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