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 the segments and then amplify the library with a single primer. From that point, she can interrogate the results with qPCR using ...
