Defixation

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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 biomarker-specific primers.

Many people are hesitant to deal with fixed tissue because it's so degraded. "You may lose some messages all together, but the ones you have retained, even in this small size, [are] not noise," Pogue-Geile remarks. "You can easily amplify things of 100 or 200 bases." Since you're just looking for correlations and not any mechanistic explanations, "that's all you need," she adds.

Cost:
Ambion's RecoverAll total nucleic acid isolation kit for formalin fixed, paraffin-embedded sections costs $5.75 per reaction, while the Rubicon TransPlex whole transcriptome amplification kit — now sold by Sigma-Aldrich — costs about $30 per reaction.