Hello everyone,
I'm having some problems with RNA isolation via TRIzol. I am working with primary human neural stem cells.
1. Usually, no RNA pellet is visible after ethanol wash, even with a large number of cells per TRIzol lysate (2.5 million cells). Keep in mind I've also tried washing the pellet once with 75% ethanol, then once with 100% ethanol.
2. Also, the 260:280 ratio is often
Hello there,
Good day to you. I have been working with Trizol myself and what I usually do is I would give thorough shaking once I introduce Trizol onto my culture flasks. Previously I just simply incubate after adding Trizol and the pellet were little. When I start to give this thorough shaking for few minutes, I could see some improvement. Hopefully this helps.
Naresh.
what I do when I want a high yield is to precipitate O/N at 4C in propanol, this allows for more RNA to precipitate. the following day just continue as normal and you will get much better yield and 260/280 values :)
RNA pellets are translucent and difficult to see. If you add glycogen as a carrier molecule during your precipitation step, a white pellet will develop which makes aspiration/resuspension much easier. Glycogen precipitates under the same conditions as RNA and does not affect down-stream applications, like cDNA synthesis. Just make sure to order guaranteed RNase-free for peace of mind.
You can also combine two protocol and use Trizol, add chloroform, spin then clean up your aqueous layer with the Qiagen RNeasy Kit.
Such as the protocol found here:
http://imageslab.fiu.edu/sites/default/files/RNA%20extraction_Trizol_RNeasy_hybrid.pdf
I have used this and find that the yields are very good and the ratios are clean. The 260/280 is almost always 1.9-2 and the 260/230 is >1.8 as long as you remove the salts correctly by adding RPE buffer.
Hi everyone - thanks for your responses. Recently, I found that I usually took more than 50% of the chloroform/TRIzol lysate volume, which probably led to phenol contamination and these readings. I also found that multiple washes with 100% ice-cold ethanol (usually, I do 5) ensures a clean pellet. Finally, I found that doing these readings in water artificially lowers the 260:280 (the readings are better after 1:1 dilution with 2X TE). With all of this, I finally achieved a 260:280 of 2.06 and 260:230 of 2.13. Problems solved!
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