How to remove salts, phenol and other contaminants from RNA samples?

Frozen_RNA_Sample_700x233-700x233.jpg

Add 1/10 vol of NH4OAc, en 2.5 volume 100% cold EtOH (100%) and 1 µl glycogen (if you think you'll the pellet without see glycogen you can do without it)

mix

Incubate 30 min at -80°C

Centrifuge 20 min at 12000g at 4°C

(remove supernatant)

Wash with 75% ice cold EtOH

Centrifuge 5 min at 12000 g at 4°C

(remove SN)

Re-suspend the pellet in DEPC (can take some time)

When extracting RNA from skin samples, you can add a Proteinase K digestion step prior to the Trizol extraction, it will increase your total RNA yield. Adding a further ethanol 75% wash at the end of the extraction will also improve a bit the overall quality of your sample, regarding the absorbance ratio.

Additionaly, check the articles by Donald Rio et al. at CSH protocols, they have useful tips and guidelines.

http://cshprotocols.cshlp.org/cgi/content/abstract/2010/6/pdb.prot5439

You could also consider using specialized kits for this kind of extraction, like the RNeasy Fibrous Tissue kit from qiagen.

http://www.qiagen.com/products/rnastabilizationpurification/rneasysystem/rneasyfibroustissuemini.aspx#Tabs=t0

Furthermore, in this report by Bruning and co-workers, they use a liquid N/mortar-and-pestle extraction with qiazol (similar acidic-buffered phenol from qiagen) followed by a cleanup step. Since they use their samples for sensitive downstream applications, could be worth to consider their method.

http://www.biomedcentral.com/content/pdf/1756-0500-4-438.pdf


Souce: NovoPro    2018-03-15