Hello,
I am working on a project where I need to recover RNA and DNA from the retina and RPE of mice (later I will likely test in rabbits and NHP's). I have been trying to find published data on the average level of nucleic acid recovery from these tissues in order to determine how efficient/inefficient my current approach is.
So far I have not found a publication listing such figures. Does anyone have personal experience or know of such a resource?
Thanks!
Hi John,
We do a lot of RNA and DNA extraction on retinas and RPE in mice in our lab and never had any trouble with the amount we got back. There is a lot of cells in retina and RPE tissues.
How much are you getting back?
Sorry, my numbers are a little approximative. From what I remember, for DNA extraction I was getting at least 50 up to 150ng/uL in 100uL elution.
For the RNA extraction, not exactly sure cause we often pool 2-3 retinas together but should be >100ng/uL (~30-50ul).
Usually, it is on the higher side if you did not damage the tissue prior to your extraction.
Hi Cécile,
Thank you for the information. I've been using Trizol previously for the RNA and DNA extractions and just received a kit from Qiagen that I am testing. I have been getting semi-variable amounts, but based on your values they look similar for RNA, although my DNA values are a bit lower with an average of about 7ug per retina which I think may be attributed to using Trizol for the extraction and isolation.
I'm hoping that the kit will provide a better yield, as well as higher quality nucleic acids. My RPE values have been much lower than the retina though, averaging about 3ug total for DNA and about 0.6ug for RNA. I've been using a bead mill to homogenize the tissue using two 30 second cycles, which I am planning on reducing since the tissue is so easy to break apart.
It's re-assuring to know that my values are around what others are getting before my next thesis advisory meeting!
Hi John,
We use the RNA and DNA extraction kit from QIAGEN. Again, there is a lot of variability with the tissue (fresh or not), the homogenization,...
The values I mentioned you are just what I remember in general but I definitely did get higher ones. As long as you use the right controls while doing PCR/RT-PCR, the amount of DNA/RNA you get should not matter too much since you are normalizing it within the samples.
Cécile Fortuny Hi Cecile, I was wondering before you extract the RNA from the retina, did you do the fixation for the eye ? Thanks
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