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Open Access Highly Accessed Methodology article

An improved method for isolation of RNA from bone

Lauren E Carter, Gail Kilroy, Jeffrey M Gimble and Z Elizabeth Floyd*

BMC Biotechnology 2012, 12:5  doi:10.1186/1472-6750-12-5

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Details of preparing the solubilized extract for RNeasy column isolation of the RNA

Elizabeth Floyd   (2012-10-24 15:40)  Pennington Biomedical Research Center email

The solubilized bone extract is separated from the bone material by centrifugation at 8,600 x g for 15 seconds at room temperature as described in the manuscript. The solubilized extract is then transferred to a new microtube and the TriReagent is removed from the solubilized extract by adding chloroform at a TriReagent:chloroform ratio of 5:1. So we add 0.2 ml chloroform to the extract and mix by inverting the tube a few times. The phases are allowed to begin separating at room temperature x 4-5 minutes before centrifuging at 12,000 x g for 15 minutes at 4C. The resulting supernatant** (aqueous phase) is transferred to a new microfuge tube and 1 volume of 70% ethanol is added to the extract before placing the extract-ethanol mixture onto the RNeasy column.

**If the supernatant has a pink color at this point, indicating the TriReagent phenol has not been completely removed, we add an additional 0.1ml chloroform and repeat the subsequent steps. This has always taken care of removing all of the TriReagent components from the extract.

Competing interests

None declared

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