Aorta RNA isolation - (Jul/25/2007 )
Hi All:
I hope that out there someone has been/is isolating total RNA from mouse aortas and can offer some help. I have attempted 3 times with extremely poor results. I have used twice the Qiagen RNA extraction kit for Fibrous Tissue, and once the TRIzol method. In 3 separate experiments with a single aorta (30-45 mg), I was able to isolate only 0.5 to 2.5 micrograms of total RNA (the higher amount with TRIzol, which does not enrich for higher MW RNA).
I have successfully isolated total RNA and mRNA from other tissues during several years in a lab, and I have experience with phenol/chloroform extraction and nucleic acid precipitation. However, I just cannot pinpoint what I might be doing wrong.
Others claim to be able to purify 14-17 micrograms of total RNA from a single mouse aorta (8-10 week old mice). With me it just does not happen. To my relief, in other publications a few say they had to use RNA carriers to concentrate the small amount of RNA obtained from a single aorta. Even doing so, the amount I obtain is not sufficient for hte set of experiments we have planned.
Does anyone have experience with this topic?
Thank you in advance! -Dory
I hope that out there someone has been/is isolating total RNA from mouse aortas and can offer some help. I have attempted 3 times with extremely poor results. I have used twice the Qiagen RNA extraction kit for Fibrous Tissue, and once the TRIzol method. In 3 separate experiments with a single aorta (30-45 mg), I was able to isolate only 0.5 to 2.5 micrograms of total RNA (the higher amount with TRIzol, which does not enrich for higher MW RNA).
I have successfully isolated total RNA and mRNA from other tissues during several years in a lab, and I have experience with phenol/chloroform extraction and nucleic acid precipitation. However, I just cannot pinpoint what I might be doing wrong.
Others claim to be able to purify 14-17 micrograms of total RNA from a single mouse aorta (8-10 week old mice). With me it just does not happen. To my relief, in other publications a few say they had to use RNA carriers to concentrate the small amount of RNA obtained from a single aorta. Even doing so, the amount I obtain is not sufficient for hte set of experiments we have planned.
Does anyone have experience with this topic?
Thank you in advance! -Dory
Hi Dory,
I used to isolate RNA from mouse and rat aortas all the time. I never used the fibrous tissue extraction protocol via Qiagen so I don't have much an opinion on that procedure. But what did work for me, was to flash freeze the cleaned aorta (removed adventitia with a dissecting microscope) in an eppendorf and then grind it up with a small mortar and pestle. When you do this, make sure that the mortar is place on dry ice to keep it cold and do the same with the pestle, or they will stick to any pieces of tissue that begin to thaw. Occasionally I would even pour a little liquid nitrogen in the mortar just before beginning. Grind the aorta up into a fine powder and then scrape it into a tube that already has 1 ml of TRIzol. Vortex and then let it sit for 10-15 minutes and then proceed with Invitrogen's protocol. I hope this helps.
Mateo
Hi Mateo:
Thank you so much for your advice. Do you think it is possible to grind the liquid nitrogen frozen aorta in an eppendorf tube with a fitting plastic pestle? I suppose it does not take too much force to break a N2 frozen tissue. Do you remember approximately the RNA yield from a single mouse aorta? Like, above 10 micrograms? Thanks again, and have a great day. -Dory
Thank you so much for your advice. Do you think it is possible to grind the liquid nitrogen frozen aorta in an eppendorf tube with a fitting plastic pestle? I suppose it does not take too much force to break a N2 frozen tissue. Do you remember approximately the RNA yield from a single mouse aorta? Like, above 10 micrograms? Thanks again, and have a great day. -Dory
Dory,
I don't know if the plastic pestle will work. The ceramic Coors ones worked best for me because you really want to grind the aorta into a fine powder. I don't remember how much I was able to isolate since this was several years ago, but it was enough for microarrays (using several micrograms).
M
Thanks a lot for your reply, Mateo. I will try using N2 soon. -Dory