Producing high quality samples

March 2005
Feature Story

William Check, PhD

The Commonwealth Technology Research Fund issued a grant three years ago to establish the Cancer Genomics Project, a collaboration between two Virginia universities—Virginia Commonwealth and George Mason—and Inova, a major health care provider in Virginia. The partnership’s focus was the development of new diagnostic tools, such as microarrays, for use in patients with cancer. This project started a tissue-acquisition system involving banking of residual tissue samples for isolation of DNA, RNA, and protein for further analysis, including gene expression microarrays. "For microarray analysis of RNA you need excellent quality material," says Catherine I. Dumur, PhD, assistant professor in the Department of Pathology at Virginia Commonwealth University. Her role in the project was to determine what processes produce high sample quality.

Of course, the first requirement is high-quality tissue, which is obtained by quick freezing after removal from the body. Moreover, tumor tissue samples should contain at least 70 percent tumor. Subsequent critical steps are RNA extraction, cDNA synthesis, and cRNA synthesis and labeling. "If we follow QC guidelines for all these steps," Dr. Dumur says, "I think microarrays would have a great chance to succeed in the clinical arena."

She offers two qualifications to this conclusion. First, microarrays are not currently used as clinical tools. "I think the main clinical impact of microarrays will be to become an ancillary tool for stratification of tumors that would be otherwise classified very similarly by current methods but that correspond to different outcomes," she says.

Second, following rigorous QC guidelines can be challenging. Having tissue frozen, rather than paraffin-embedded, demands special attention. The Cancer Genomics Project recruited dedicated tissue-acquisition coordinators who can access the OR schedule and transport freshly removed tissue to the pathologist for evaluation and snap freezing. Time from removal of tissue to freezing should be recorded and kept to a minimum, Dr. Dumur says. In the Cancer Genomics Project that time is usually about 20 minutes.

Tumor cells from samples containing less than 70 percent tumor may have to be enriched by laser capture microdissection, which may affect gene expression results when compared with non-microdissected samples. Also, samples with very small amounts of RNA require linear amplification, which can introduce bias. Dr. Dumur extracts RNA with TRIzol followed by a Qiagen cleanup process to further remove inhibitors. She advocates QC analysis after RNA extraction, cDNA synthesis, and cRNA labeling, and these QC criteria were published recently (Dumur CI, et al. Clin Chem. 2004;50:1994-2002).

Much, however, remains to be done. Microarray data acquired by different laboratories from the same tissue samples have to be analyzed to test interlaboratory reproducibility, "similar to proficiency testing," Dr. Dumur says, and ensure the quality of the overall process.