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Real-time quantitative reverse transcription-PCR assay for renal cell carcinoma-associated antigen G250
Ye Chuanzhong1, Guan Ming, Zhang Fanglin
1Department of Urology, Zhong shan Hospital, Medical Center of Fudan University, 180 Feng lin Road, Shanghai 200032, People's Republic of China. chuanzhong@mycity.com.cn
Objectives:
Gene amplification/expression of G250 is a major event in human renal tumorigenesis. G250-based therapeutic agents and G250-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen G250 alterations in renal cell cancer (RCC) patients and investigate the relationship between G250 mRNA expression and RCC.
Methods:
We developed a quantitative RT-PCR assay for the measurement of G250 mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The method has been applied to the measurement of quantitative mRNA level of G250 in 31 cases RCC and 6 normal renal tissues.
Results:
The dynamic range was 10(3)-10(8). The relationship between Ct and log starting concentration was linear (r=0.99). G250 expression was present in all RCCs with G250 amplification but was absent in normal ones. G250 mRNA expression ranged from 2.9 x 10(3) to 6.5 x 10(7) copy/microg RNA, with a mean value of 3.5 x 10(6) copy/microg RNA. The expression of G250 revealed an inverse correlation to tumor grade. G250 mRNA level did not correlate with the cell types and clinical stages (P>0.05).
Conclusions:
G250 has the potential to be used as a marker of diagnosis and increasing proliferation in RCC. This new simple, rapid, semi-automated assay was a major alternative to competitive PCR and Northern blot analysis for gene alteration analysis in human tumors and might be a powerful tool for large randomized, prospective cooperative group trials and supporting future G250-based biological and gene therapy approaches.
Insights
A new quantitative RT-PCR assay accurately measures G250 mRNA expression in renal cell cancer (RCC). This method can identify G250 alterations and supports G250-based therapies for kidney cancer.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Gene amplification and expression of G250 are critical in renal cell cancer (RCC) development.
- G250-targeted therapies and gene therapy strategies are emerging for RCC treatment.
- Accurate methods are needed to screen for G250 alterations and understand its role in RCC.
Purpose of the Study:
- To develop and validate a sensitive quantitative RT-PCR assay for measuring G250 mRNA expression in RCC.
- To investigate the correlation between G250 mRNA levels and clinicopathological features of RCC.
- To assess the utility of G250 as a diagnostic and prognostic marker in RCC.
Main Methods:
- A quantitative real-time RT-PCR assay was developed using fluorogenic probes and the ABI PRISM 7700 Sequence Detector System.
- The assay measured G250 mRNA levels in 31 RCC tissues and 6 normal renal tissues.
- Assay validation included dynamic range determination and linearity assessment (r=0.99).
Main Results:
- The quantitative RT-PCR assay demonstrated a dynamic range of 10(3)-10(8) with excellent linearity.
- G250 mRNA was detected in all RCCs with G250 amplification but was absent in normal tissues.
- G250 expression inversely correlated with tumor grade, but not with cell type or clinical stage.
Conclusions:
- The developed RT-PCR assay is a sensitive, rapid, and semi-automated tool for analyzing G250 gene alterations in human tumors.
- G250 mRNA expression shows potential as a diagnostic marker and indicator of proliferation in RCC.
- This assay can support G250-based therapeutic strategies and large-scale clinical trials for RCC.