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There are two tumor samples floating in a 10% formaldehyde solution (i.e formalin). Is there a protocol for RNA extraction under these circumstances? I am concerned that using the protocol for Formalin Fixed Paraffin Embedded (FFPE) tissue will damage the RNA, since there is no paraffin for the xylene to remove.

If I were to use the FFPE protocol without xylene, should I homogenize the tissue before removing the formalin, or after?

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  • $\begingroup$ Can you please link to or describe in detail this FFPE protocol that you're referring to? $\endgroup$ – MattDMo Jul 6 '16 at 1:08
  • $\begingroup$ @MattDMo It is the RNeasy FFPE Kit mentioned in WYSIWYG's answer. $\endgroup$ – Arthur Miller Jul 6 '16 at 17:52
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Formaldehyde forms adducts with RNA and can also make it more susceptible to degradation. It can also cause crosslinks to form between RNA and proteins (or possibly other RNAs). For all these reasons extraction of high quality RNA from formaldehyde fixed samples is very difficult and the yields are low (Howat and Wilson, 2014).

Have a look at this article by Evers et al. (2011) which explores optimal conditions for removal of the formaldehyde adducts. However, this study does not involve actual RNA extraction from fixed tissues, which would be even more difficult.

RNeasy FFPE Kit from Qiagen claims to be specially designed for RNA extraction from formaldehyde fixed samples. The product description says that the lysis buffer contains substances that reverse the modifications formed because of formaldehyde. There is a similar kit from Thermo-Fisher. Russel et al. (2013), report that the RNA yield was similar between fixed (using Qiagen RNeasy FFPE kit) and unfixed samples (using Qiagen RNeasy Plus mini Total RNA extraction kit).

should I homogenize the tissue before removing the formalin, or after?

Homogenize after removing formalin. Since the tissues are already brittle because of formaldehyde fixation, you need not use harsh homogenization procedures. Pipette gently. You can use proteinase-K and DNAse to remove proteins and DNA respectively (RNeasy kit involves these steps).

For future experiments, you can store the tissues in RNAlater solution (Thermo-Fisher; or similar formulations from other vendors/lab). Store one part of tissue in formaldehyde for IHC/ISH and the other in RNAlater for RNA/DNA extraction.

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  • $\begingroup$ If I recall correctly, the RNeasy FFPE kit is designed for extracting RNA from formalin-fixed, paraffin-embedded tissues; however, I've heard that it works fine for just formalin-fixed tissues, skipping the deparaffinization step. I haven't tried it myself, though $\endgroup$ – iayork Jul 6 '16 at 11:46

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