I am looking for recommendations on a kit to extract RNA from whole blood from a songbird species. We cannot take a lot of blood from the birds since they are small. Are there kits that allow for ~100-200 ul blood samples? If not, do people have other recommendations?
Hi Joel, if you haven't received an answer, my suggestion would be a Qiagen kit. You can check with my lab to see which is the best bet. Good luck.
Hi Joel,
I have just extracted very good quality RNA from passerine blood stored in tempus. I use 100ul of whole blood.
If you extract the RNA using trizol, then treat the RNA in solution with DNase 1 solution provided in the qiagen RNeasy micro kit, then pass it through the spin columns provided with the micro kit as per manufacturer instructions this should yield good results.
I tried the kit on its own but I get much higher yields with TRIzol. The nano drop 260/230 ratio will be low, but this is because of the guanidine thayacyanate they put in their lysis buffers which does not inhibit down stream applications. If you look at the RNA using the bioanalyser, it is good quality using this method and qubit yields are good for qPCR/RNAseq for instance.
Hi Daisy,
Thank you so much for the advice. What's the ratio of TRIzol to blood? Can it be stored in a 1.5ml sterile microcentrifuge tube? Do you recommend immediate treatment of DNase 1 right after placing the blood in TRIzol? I am looking for a method where I can store the samples easily, as they will be collected in the field.
Best,
Joel
Hi Joel,
The ratio I used was 100ul of blood to 1ml of trizol.
We collected 250ul of blood in tubes containing tempus storage reagent, although I would not recommend the tempus reagent due to the way our blood has coagulated, making it difficult to separate RBCs from plasma etc. I would either store it in TRIzol directly, or store in RNA later if we were to do it again.
The DNase treatment isn't done until after you've done your trizol extraction (removed the chloroform layer, precipitates with isopropanol, washed pellet in ethanol and dried). Trizol protocols are very similar and can be found online.
Best wishes,
Daisy
If I were to use RNAlater (which is a bit safter than TRIzol in the field) for storage, what would you think is a good ratio of blood to RNAlater?
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