I've been working on improving RNA quality lately since I've had a number of bad qPCR results in the past few months. We use the Trizol method in our lab, and recently there has been a lot of precipitation during the RNA isolation. Brief rundown, we homogenize our tissues (I'm using mouse hearts and fats) in Trizol per manufacturer's instructions, followed by a spin to remove insoluble material, and then an addition of chloroform to isolate the RNA. After spinning and removing the aqueous phase, we add it to isopropanol. At this stage, my samples turn extremely cloudy, obviously containing some kind of precipitation. This precipitate can be spun down, and initially forms a gel-like pellet which turns opaque white after washing with 70% EtOH. There is not supposed to be cloudiness at this stage though...the solution is supposed to turn clear after adding the aqueous phase.
I tried this with a blank set of samples (i.e. Trizol without homogenized tissue) and I got the same result...the aqueous phase I pulled off of the blank becomes cloudy after adding to iso, and can be spun down to produce a pellet that looks just like what came out of my tissue samples. I should also note that in my tissue samples, I could see a tiny blue dot after the first EtOH wash (we use glyco blue to precipitate with RNA), so there was definitely a good RNA pellet in there, but it was surrounded by a big flaky piece of white precipitate. The A260/230 ratio is also very low with RNA I get from these cloudy samples, usually around 0.1 (260/280 is good at around 2.0).
Has anyone else had this issue before? Is this some kind of salt precipitation or something else, and how should I get rid of it? I'm guessing maybe the Trizol got contaminated since it happened with blank samples (and I used fresh chloroform and iso, and adding chloroform straight to iso without Trizol did not form this cloudy precipitation), but before I throw the bottle out I'd like to see if this is a known issue I can fix. Any advice is appreciated!
Hi Royer, I guess your homogenization process might not be perfect. If you want to isolate RNA from white adipose tissue, I would recommend you to use 200mg of tissue in 1ml of QIAzol Lysis Reagent. However for heart you can use 20mg in 1ml QIAzol Lysis Reagent, which is sufficiently give RNA for the 100qPCR. If you are isolating RNA from the FAT tissue you would certainly see white precipitation which could be FAT or protein. Let me know if you need any further help.
I would recommend you to use Tissue raptor for homogenization (https://www.qiagen.com/us/shop/automated-solutions/sample-disruption/tissueruptor-ii/#orderinginformation).
I process more than 1000 samples for the RNA isolation from different mouse organs such Liver, kidney, white and brown adipose tissue, spleen, brain, heart etc,
Very important point Homogenization should be done in 1min. once it homogenize immediately transfer to the Dry Ice, you can keep your sample upto a week. during homogenization keep your sample at 4oC.
Let me know if you need further help.
Thank you
Hi Charlotte
I recommend using 100 mg of powdered tissue in mortar with liquid nitrogen and then adding the Trizol.
Hi.. I used trizol reagent as well..during phase separatio (add chloroform and centrifuge, the sample separated and the upper phase is colourless clear aqueous phase. however, after rna precipitation (add cold isopropyl alcohol and centrifuge), there’s a brown pellet on all sample that I extracted.. I extracted five sample.. is there anything i should do? Us it okay to add glycogen? My sample is classified as tissue samples (pooled mosquitoes).. any advice and suggestion are highly appreciated..
thank you for your help..
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