Technology for automated, rapid, and quantitative PCR or reverse transcription-PCR clinical testing.
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 approximately 35 min.


METHODS
Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-microm 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 approximately 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 deltaCt for a new measurement was +/-1.54 cycles, and for breast cancer samples, the interval for a newly observed deltaCt 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 approximately 35 min and could therefore be used for intraoperative testing when applicable.
Raja
Siva
S
Department of Surgery, Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Ching
Jesus
J
Xi
Liqiang
L
Hughes
Steven J
SJ
Chang
Ronald
R
Wong
Wendy
W
McMillan
William
W
Gooding
William E
WE
McCarty
Kenneth S
KS
Chestney
Melissa
M
Luketich
James D
JD
Godfrey
Tony E
TE
D000230:Adenocarcinoma / Q000737:chemistry / Q000175:diagnosis / Q000473:pathology; D001322:Autoanalysis; D001943:Breast Neoplasms / Q000737:chemistry / Q000175:diagnosis / Q000473:pathology; D005260:Female; D005453:Fluorescence; D005629:Frozen Sections; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000175:diagnosis / Q000473:pathology; D008207:Lymphatic Metastasis; D008545:Melanoma / Q000737:chemistry / Q000175:diagnosis / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D011237:Predictive Value of Tests; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods
D012313:RNA