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Clinical Chemistry 51: 882-890, 2005. First published March 3, 2005; 10.1373/clinchem.2004.046474
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Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 2005;51:882-890.)
© 2005 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Technology for Automated, Rapid, and Quantitative PCR or Reverse Transcription-PCR Clinical Testing

Siva Raja1, Jesus Ching2, Liqiang Xi1,1, Steven J. Hughes1, Ronald Chang2, Wendy Wong2, William McMillan2, William E. Gooding3, Kenneth S. McCarty, Jr4, Melissa Chestney1, James D. Luketich1 and Tony E. Godfrey1,1,a

Departments of1 Surgery, 3 Biostatistics, and 4 Pathology, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213.
2 Cepheid, Sunnyvale, CA 94089.

aAddress correspondence to this author at: Mount Sinai School of Medicine, One Gustave L. Levy Place, East Building, Room 1070C, New York, NY 10029. Fax 212-828-4221; e-mail tony.godfrey{at}mssm.edu.

Background: PCR-based assays can improve clinical care, but they remain technically demanding and labor-intensive. We describe a new instrument, the GeneXpert®, that performs automated nucleic acid isolation, reverse transcription, and fluorescence-based quantitative PCR in ~35 min.

Methods: Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-µm frozen tissue sections). The reproducibility of automated RNA isolation, reverse transcription, and quantitative PCR was determined by replicate (n = 10) analysis of 10 tissues, using duplex (target and endogenous control) reverse transcription-PCR reactions for two gene combinations. The GeneXpert was then used to perform rapid analysis of lymph nodes from melanoma, breast cancer, and lung cancer patients and analysis of melanoma metastatic to the lung, primary lung adenocarcinoma, and healthy lung tissue.

Results: On the GeneXpert, RNA was recovered in slightly over 6 min, and the yield was ~70% of that from parallel Qiagen reactions. The RNA integrity was comparable to that of Qiagen-isolated RNA as determined by gel electrophoresis. For the melanoma samples, the 95% prediction interval for the {Delta}Ct for a new measurement was ±1.54 cycles, and for breast cancer samples, the interval for a newly observed {Delta}Ct was ±1.40 cycles. GeneXpert assays successfully detected the presence of metastatic melanoma, breast cancer, and lung cancer in lymph nodes and also differentiated among metastatic melanoma, lung adenocarcinoma, and healthy lung.

Conclusions: RNA yield and integrity on the GeneXpert are comparable to benchtop methods. Reproducibility of the GeneXpert data is similar to that seen with manual methods in our hands but may need improvement for some applications. The GeneXpert can perform RNA isolation, reverse transcription, and quantitative PCR in ~35 min and could therefore be used for intraoperative testing when applicable.




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