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A novel method for real time quantitative RT-PCR.
Open accessAbstract
A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.
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Cited by
A new mathematical model for relative quantification in real-time RT-PCR
Pfaffl, M. W. (2001) · Nucleic Acids Research · doi:10.1093/nar/29.9.e45 · Cited by 34,610
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Nolan, T., Hands, R. E., & Bustin, S. A. (2006) · Nature Protocols · doi:10.1038/nprot.2006.236 · Cited by 2,247
Choi, H. M. T., Schwarzkopf, M., Fornace, M. E., Acharya, A., Artavanis, G., Stegmaier, J., Cunha, A., & Pierce, N. A. (2018) · Development · doi:10.1242/dev.165753 · Cited by 1,495
Twenty-Five Years of Quantitative PCR for Gene Expression Analysis
VanGuilder, H. D., Vrana, K. E., & Freeman, W. M. (2008) · BioTechniques · doi:10.2144/000112776 · Cited by 1,246
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Gerber, H.-P., Hillan, K. J., Ryan, A. M., Kowalski, J., Keller, G.-A., Rangell, L., Wright, B. D., Radtke, F., Aguet, M., & Ferrara, N. (1999) · Development · doi:10.1242/dev.126.6.1149 · Cited by 915
Metadata
- Authors
- Gibson, U. E., Heid, C. A., & Williams, P. M.
- Venue
- Genome Research
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 1996-10
- Volume
- 6
- Issue
- 10
- Pages
- 995-1001
- ISSN
- 1088-9051 (ISSN-L)
- OpenAlex
- W2145684092
- Cited by
- 2,075
- References
- 0
- Type
- posted content
- Language
- en
- Open access
- bronze
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- Gibson, U. E., Heid, C. A., & Williams, P. M. (1996). A novel method for real time quantitative RT-PCR. Genome Research. https://doi.org/10.1101/gr.6.10.995
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