Defixation

Credit: © CNRI / Photo Researchers, Inc." /> Credit: © CNRI / Photo Researchers, Inc. User: Kay Pogue-Geile, National Surgical Adjuvant Breast and Bowel Project, Pittsburgh, Pa. Project: Developing prognostic profiles for breast cancer using tumor samples obtained from clinical trials. Problem: Nucleic acids are degraded and modified by the

Josh P. Roberts
Apr 1, 2008
<figcaption> Credit: © CNRI / Photo Researchers, Inc.</figcaption>
Credit: © CNRI / Photo Researchers, Inc.

User:
Kay Pogue-Geile, National Surgical Adjuvant Breast and Bowel Project, Pittsburgh, Pa.

Project:
Developing prognostic profiles for breast cancer using tumor samples obtained from clinical trials.

Problem:
Nucleic acids are degraded and modified by the formalin fixation process used in paraffin-embedded tissue.

Solution:
Treating embedded sections with xylene gets rid of the paraffin, and heating can remove most cross-linked proteins. You can use commercial kits, such as those available from Roche or Ambion, to clean away the formalin and extract the RNA.

After these treatments only 100- to 200-base fragments remain, some with short poly-A tails, and some without. Traditional reverse-transcription using an oligo-dT primer won't work very well on such degraded RNA. So, Pogue-Geile uses a kit from Rubicon Genomics to add adapters to both ends of...