Temperature-controlled primer limit for multiplexing of rapid, quantitative reverse transcription-PCR assays: application to intraoperative cancer diagnostics.
Clin Chem
; 48(8): 1329-37, 2002 Aug.
Article
en En
| MEDLINE
| ID: mdl-12142391
ABSTRACT
BACKGROUND:
Rapid-cycling, real-time PCR instruments bring the opportunity for improved intraoperative detection of metastasis to sentinel lymph nodes. Rapid, standardized, and internally controlled assays need to be developed that are sensitive and accurate.METHODS:
We describe rapid, multiplexed, internally controlled, quantitative reverse transcription-PCR (QRT-PCR) assays for tyrosinase and carcinoembryonic antigen mRNAs on the SmartCycler (Cepheid). We used a temperature-controlled primer-limiting approach to eliminate amplification of the endogenous control gene as soon as its signal had reached threshold. Positive-control oligonucleotide mimics were incorporated into all reactions to differentiate failed reactions from true negative samples.RESULTS:
The optimized assays for rapid QRT-PCR yielded results with threshold cycle values that were only 1-2 cycles higher than slower, more conventional protocols. In rapid PCR, the temperature-controlled multiplex assay was quantitative over a dynamic range of at least 15 cycles, compared with only 6 cycles for conventional multiplexing methods. All histologically positive lymph nodes examined were also QRT-PCR positive for the appropriate marker, and the exogenous, internal positive-control mimics produced signals in all negative samples.CONCLUSION:
Internally controlled, rapid QRT-PCR assays can be performed in an intraoperative time frame and with sufficient sensitivity to detect histologically identified metastases to lymph nodes.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
/
Ganglios Linfáticos
/
Metástasis Linfática
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Female
/
Humans
Idioma:
En
Revista:
Clin Chem
Asunto de la revista:
QUIMICA CLINICA
Año:
2002
Tipo del documento:
Article
País de afiliación:
Estados Unidos