I am using the miRCURY kit of Exiqon for small RNA isolation from crude plasma and, as instructed, I add 1 µg of MS2 carrier RNA in order to improve the yield.
Given the fact that, in most extractions done homogeneously I always get a final concentration of approx. 20 ng/µl, I thought that the presence of the same amount of carrier RNA was responsible. Today I realized that, when putting the same amount of MS2 in sterile PBS, the result in the Nanodrop is not the same, as RNA is hardly detected.
Does this finding mean that the RNA concentration in the eluates corresponds to small RNAs, as the MS2 remains in the column?
I know that its length is 3569 nt so theoretically it should not be able to pass through.
Hi Nikiforos
I am pretty familiar with Exiqon biofluids kits and I have to tell you that I never used MS2 as carrier. You basically do not need it. The carrier is cool to be used in other kind of experiments like RNA-IP.
Anyhow, if you really like to use the carrier, you must be aware that you will get it always during purification. RNA micro-spin columns are not size-exclusion columns, but ionic-exchangers. The purification and cleaning process by the column is based on the selective binding of negative molecules (RNA) to a anion-exchange resin (embebed in the filter of the spin-column) under slightly hydrophobic conditions (you must add ethanol or isopropanol to your sample before loading the column). So, you have no way to get rid of the MS2 RNA by using this protocol.
Your results are probably due to some technical artifact, but in principle the MS2 RNA should also bind to the purification micro-spin column. Be sure that you performed the protocol exactly in the same way that you did it for plasma, but substituting the plasma by your PBS-MS2 solution.
I would advice you to give a try without the carrier. And do not forget that using Nanodrop to quantify RNA coming from plasma samples is useless because of the sensitivity thresold of the machine. You need to use more sensitive devices as Qubit or Bioanalyzer.
Best of luck.
Paco
Thank you very much for your great answer Paco!! I have been using this technique for 3,5 years now, without any problem. I always put 1 µg of MS2 carrier and I have around 20 ng/µl of small RNA concentration on Nanodrop. As I thought that this was a result of the MS2 in the eluate, I tested it alone in PBS and it was not what I expected as it was approaching 0. On the other hand, addition of spike-ins with the MS2 in the PBS slightly increased the yield. Plasma alone without MS2 or spike-ins only yielded 3-4 ng/µl.
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