Samples were homogenized with a mortar and pestle and then passed through a syringe but at the first centrifuge step the sample is getting stuck - we can't see anything on the membrane and only about half is coming through.
Has anyone got any experience with this kind of tissue and these columns? Thanks!
Helen Marshall
In my experience with the RNeasy kits it is usually the genomic DNA that won't get sheared, which interferes with the RNA extraction and results in low RNA elution.
(But, loading too much tissue relative to lysis buffer can also result in poor RNA capture so you might want to review this as well.)
I haven't done this type of extraction with a mortar and pestle. I have used a bead homogenizer for small volumes (30 mg) tissue or a dounce homogenizer. I found the bead homogenizer the best for fully disrupting the cells and also shearing the gDNA so it doesn't interfere with the RNA extraction.
If you stay with the mortar and pestle and are using the syringe to then further break up the cells. What size syringe matters! Progressive smaller bore size needles work the best: start with 16 G (10x), 21G (10x) and then 30G (10x) to shear the DNA and do the last homogenization. You should then be able to pellet and see a very small white pellet at the bottom after the centrifugation step and the solution should be just like water (not gelatinous).
Good luck!
Lisa Bradley thanks so much, we really appreciate that! We'll give that a go.
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