Rapid, quantitative reverse transcriptase-polymerase chain reaction: Application to intraoperative molecular detection of occult metastases in esophageal cancer

Raja, Siva; Luketich, James D.; Kelly, Lori A.; Gooding, William E.; Finkelstein, Sydney; Godfrey, Tony E.

doi:10.1067/mtc.2002.119884

Cited by 22 publications

(23 citation statements)

References 22 publications

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“…These micrometastatic deposits can be detected with immunohistochemical analysis using monoclonal antibodies to stain tumour-specific antigens or using Polymerase Chain reaction (PCR) at the molecular level [37][38][39][40][41][42][43][44]. It is unclear whether these nodal micrometastases represent tumour cells with metastatic potential or just shredded cells, which will be cleared by the immune system or remain dormant in a non-proliferating phase [39,45].…”

Section: Micrometastases In Lymph Nodesmentioning

confidence: 99%

Lymph node metastases and prognosis in oesophageal carcinoma – A systematic review

Kayani

Zacharakis

Ahmed

et al. 2011

European Journal of Surgical Oncology (EJSO)

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Oesophageal cancer is the seventh most common cause of cancer-related death in the developed world and the incidence of oesophageal adenocarcinoma is now the fastest growing of any gastrointestinal cancer. Lymph node involvement is the single most important prognostic factor in oesophageal cancer. Imaging to determine the extent of lymph node involvement and plan treatment often requires a combination of modalities to avoid under-staging. The seventh edition of the staging system released by the International Union Against Cancer (IUCC) has stratified lymph node involvement according to the number of lymph nodes involved and redefined its groupings for location of metastatic lymph node involvement. This review discusses the prognostic and treatment implications of these modifications and explores micrometastatic lymph node involvement, capsular infiltration and lymph node ratio as possible additions to the staging system.

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Section: Micrometastases In Lymph Nodesmentioning

confidence: 99%

Lymph node metastases and prognosis in oesophageal carcinoma – A systematic review

Kayani

Zacharakis

Ahmed

et al. 2011

European Journal of Surgical Oncology (EJSO)

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“…Serum RNA was also analyzed for the presence of MART-1 and tyrosinase in a mutiplexed rapid QRT reactions on the SmartCycler instrument (Cepheid) according to previously published protocols (29 ). Total RNA (from 3 mL of serum) was reverse-transcribed by gene-specific reverse transcription primers at 48°C for 20 min.…”

Section: Real-time Qrt-pcrmentioning

confidence: 99%

Characterization of Amplifiable, Circulating RNA in Plasma and Its Potential as a Tool for Cancer Diagnostics

El-Hefnawy¹,

Raja²,

Kelly³

et al. 2004

Clinical Chemistry

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244

179

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Background: Several recent reports have described the detection of circulating, cancer-related RNA molecules in serum or plasma from cancer patients, but little is known about the biology of this extracellular RNA. We aimed to determine how RNA is protected against degradation in serum, to optimize RNA isolation from large volumes of serum, and to test our optimized assays for serum-based cancer detection. Methods:We used quantitative reverse transcription-PCR (QRT-PCR) analysis to investigate the isolation and biology of extracellular plasma RNA. We then examined the presence of amplifiable RNA transcripts in plasma and serum from controls and from patients with esophageal cancer and malignant melanoma. Results: We found that extracellular RNA in plasma is highly degraded and can be isolated most efficiently by guanidinium-phenol extraction followed by precipitation. Extracellular RNA is stable in serum for up to 3 h but is destroyed immediately by addition of detergents. Extracellular RNA can be captured on 0.2 m filters, allowing concentration of RNA from several milliliters of plasma. When we concentrated RNA from up to 4 mL of serum, detection of cancer-related transcripts in serum from cancer patients and controls was infrequent and inconsistent. Conclusions: Extracellular RNA is most likely protected within protein or lipid vesicles, possibly apoptotic bodies, which can be disrupted by detergents. Despite

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“…When combined with our previously described methods for rapid reverse transcription and quantitative PCR multiplexing (7,10,11 ), QRT-PCR assays can be completed in Ͻ35 min from tissue to result. With this capability comes, for the first time, the potential to perform molecular diagnostic assays in rapid, point-of-care scenarios.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to providing quantitative data, real-time PCR has, in most cases, eliminated the need for post-PCR processing for detection of products, thereby greatly reducing assay time and minimizing, if not eliminating, the possibility of contamination and false-positive results. Other technical advances also allow the thermal cycling portion of realtime PCR assays to be performed extremely rapidly, with results in 20 min or less (5)(6)(7). As a result, real-time PCR assays are beginning to enter clinical testing in situations in which rapid, point-of-care diagnostics are required, such as peripartum testing for group B streptococcus in pregnant women (8,9 ).…”

mentioning

confidence: 99%

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

Raja

Ching²,

Xi³

et al. 2005

Clinical Chemistry

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119

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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 ϳ35 min. Methods: Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-m 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 ϳ70% 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 ⌬Ct for a new measurement was ؎1.54 cycles, and for breast cancer samples, the interval for a newly observed ⌬Ct was ؎1.40 cycles.

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Rapid, quantitative reverse transcriptase-polymerase chain reaction: Application to intraoperative molecular detection of occult metastases in esophageal cancer

Cited by 22 publications

References 22 publications

Lymph node metastases and prognosis in oesophageal carcinoma – A systematic review

Lymph node metastases and prognosis in oesophageal carcinoma – A systematic review

Characterization of Amplifiable, Circulating RNA in Plasma and Its Potential as a Tool for Cancer Diagnostics

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

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