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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. |
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