I want to isolate mRNA from bird blood (nucleated blood cells), but haven't found a protocol that does that directly. Most of the protocols I've seen for blood entail a two step process: isolating total RNA, and then enriching for mRNA. On the other hand, there are non-blood protocols that can isolate mRNA directly. Does anyone know of a one-step protocol for getting mRNA from whole blood?
Hi, I do not think that such a kit exists. Why don't you extract total RNA and afterwards mRNA, it will take you not so long.Good luck!I
Yes, the two-step process is my backup plan; however, for reasons of both cost-effectiveness and obtaining optimal yield, I would prefer to use a one-step mRNA isolation. I have not found a commercial kit, but I am interested in whether anyone has developed a protocol in their lab, modified from kits or other protocols.
I am not aware of any protocol that could achieve that. Perhaps the way mRNA estraction works (as of now, at least) makes it impossible. I found a partial explanation in a kit's protocol: "This kit is optimized to select poly(A) RNA from total RNA in a very low ionic-strength buffer and it cannot be used with crude cell or tissue lysates." (Ambion polyA purist kit)
In case it is of interest, I found a kit which will isolate mRNA from whole blood (and other tissue types). I have not tried it yet and am not sure about the blood storage conditions (it seems that the protocol uses blood that is frozen but not stored in a buffer like RNAlater or Trizol, which might lead to RNA degradation). Nonetheless, it is worth a try for me: https://www.lifetechnologies.com/order/catalog/product/K157002?ICID=cvc-mrna-extraction-c4t1
Thanks Loren! I am also in search of a good protocol to extract mRNA from avian blood
Jamie (and all), I have had the most success with the Dynabeads mRNA Direct kit, with some modifications. I tend to get just as much (and as high-quality) mRNA as when I extract Total RNA and then enrich for mRNA, and it's a lot faster. Here's the kit: https://www.thermofisher.com/order/catalog/product/61012
It's pricey but I modify the amounts, something like 100ul of beads for 20uL blood, 400ul of lysis buffer, 500ul of buffer A, and 200-300ul of buffer B (it runs out first). Also, I think the protocol (as with others) calls for passing the blood through a syringe. This was not even close to being possible for me. Instead, I vortexed the blood and lysis buffer to lyse the cells. I used a vortex with a head that could hold lots of tubes and vortexed for ~15-30 minutes.
Good luck!
Great, thanks again Loren! Your post was really helpful and I might consider the Dynabeads kit.
Can I ask, what species of bird were you working with?
I'm trying to design a pilot study to look at gene expression in heat stressed penguins
Ooh, interesting! I am working primarily with the Hawaii amakihi, a species of honeycreeper. They are small and so I am limited in how much blood I can collect (~50uL max for RNA, as I have to save some for DNA). If you can collect more blood, it would be easier for you to optimize the amount to use in each extraction and how much of each of the buffers...
This info was incredibly useful as I am testing direct mRNA isolation, thanks so much for providing info about your experience!
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