we use trazol kit for extrcting RNA from sputum ;although we change our extraction kit we faced low 260/280 ratio of nanodrop.(1.5)
according to well-known contamination of sputum (carbohydrate);
what is the best solution for obviating contamination with less RNA destruction?
Dear Ahmad,
I've worked with RNA from plant cell cultures, with long amounts of carbohydrates and polyphenols, which produce interference with RNA extraction. I recommend you that use some sequestring agents in your extraction buffer, like PVPP (Polyvinil(poly)pirrolydone at 2%. PVPP is water insoluble, but can be easily removed from your RNA sample by centrifugation. Other option is carry out the RNA precipitation in two differents steps, one with 2-propanol and Sodium acetate 3M pH5.2, and other with Lithium Chloride 10M. (Morante-Carriel et al., 2014).
Good luck!
you should use fresh sample if possible. try to use a protcol fromy any papers working on your RNA specially it use proteinase K
I have very good success using Qiagen's QiaAmp RNA extraction kits for RNA from a wide variety of sources. These kits give good yields of very clean RNA. You may want to try that instead. I personally don't like working with phenol-based extraction methods if I can avoid it.
Hi Ahmad
I also use trizol for my RNA extractions, and then the old school solvent precipitation method. Not sure how the steps are in the modern kits, but when I am separating my aqueous form organic phase by centrifugation steps, I always make sure I centrifuge extra time (always at 4 degrees), so my RNA is completely separated from organic and DNA phase. Meaning, the more transparent the better. I also make sure to leave the final 100 uL of RNA phase in the tube. You will lose a bit of sample, but you will win in RNA quality. In addition, I use this kit to remove DNA contaminations and always gives us very good results in the nanodrop https://www.thermofisher.com/order/catalog/product/AM1906
good luck!!
Hi, Ahmad.
I do agree with other scientists around. Take a look at this two papers. It may help you a litlle bit.
https://erj.ersjournals.com/content/44/Suppl_58/P995
Am J Respir Cell Mol Biol. 2017 Nov;57(5):626-628. doi: 10.1165/rcmb.2017-0198LE.
Improving Gene-Expression Studies from Sputum: A Multistep Optimization of RNA Isolation and qPCR Protocols.
Good luck.
Hi
You can use trizol twice first time use two tubes of the same sample and when you finish collect the two tubes in one tube and use trizol again for the second time
Elias Hurtado-Gaitán Hurtado-Gaitán tnx for answering ; what volume of LiCl is needed ?
Hi!
You just need to precipitate the RNA with 0.1 volume of 3 M sodium acetate and 1 volume of isopropanol or 2.5 volumes of cold 96% ethanol at -20 degrees for 1-2 h or at -80 dergrees for 30 min. Then centrifuge, aspirate the ethanol, wash pellet twice with 70% ethanol, dry and dissolve in nuclease free water.
Best regards
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