Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction
Quantification of mRNA expression levels using real-time reverse transcription PCR (RT-PCR) is increasingly used to validate results of DNA microarrays or GeneChips®. It requires an improved method that is more robust and more suitable for high-throughput measurements. In this report, we compare a u...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2005-02-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/05382RR05 |
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| author | Van Luu-The Nathalie Paquet Ezequiel Calvo Jean Cumps |
| author_facet | Van Luu-The Nathalie Paquet Ezequiel Calvo Jean Cumps |
| author_sort | Van Luu-The |
| collection | DOAJ |
| description | Quantification of mRNA expression levels using real-time reverse transcription PCR (RT-PCR) is increasingly used to validate results of DNA microarrays or GeneChips®. It requires an improved method that is more robust and more suitable for high-throughput measurements. In this report, we compare a user non-influent, second derivative method with that of a user influent, fit point method that is widely used in the literature. We also describe the advantage of using a double correction: one correction using the expression levels of a housekeeping gene of an experiment as an internal standard and a second using reference expression levels of the same housekeeping gene in the tissue or cells. The first correction permits one to decrease errors due to sample preparation and handling, while the second correction permits one to avoid the variation of the results with the variability of housekeeping in each tissue, especially in experiments using various treatments. The data indicate that the real-time PCR method is highly efficient with an efficiency coefficient close to the theoretical value of two. The results also show that the second derivative method is more accurate than the fit point method in quantifying low gene expression levels. Using triplicate experiments, we show that measurement variations using our method are low with a mean of variation coefficients of <1%. |
| format | Article |
| id | doaj-art-d24ac87e2d5a449697ee41559413cd9e |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2005-02-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-d24ac87e2d5a449697ee41559413cd9e2025-08-20T02:25:36ZengTaylor & Francis GroupBioTechniques0736-62051940-98182005-02-0138228729310.2144/05382RR05Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correctionVan Luu-The0Nathalie Paquet1Ezequiel Calvo2Jean Cumps31Laval University, Quebec, Canada1Laval University, Quebec, Canada1Laval University, Quebec, Canada2University of Louvain, Bruxelles, BelgiumQuantification of mRNA expression levels using real-time reverse transcription PCR (RT-PCR) is increasingly used to validate results of DNA microarrays or GeneChips®. It requires an improved method that is more robust and more suitable for high-throughput measurements. In this report, we compare a user non-influent, second derivative method with that of a user influent, fit point method that is widely used in the literature. We also describe the advantage of using a double correction: one correction using the expression levels of a housekeeping gene of an experiment as an internal standard and a second using reference expression levels of the same housekeeping gene in the tissue or cells. The first correction permits one to decrease errors due to sample preparation and handling, while the second correction permits one to avoid the variation of the results with the variability of housekeeping in each tissue, especially in experiments using various treatments. The data indicate that the real-time PCR method is highly efficient with an efficiency coefficient close to the theoretical value of two. The results also show that the second derivative method is more accurate than the fit point method in quantifying low gene expression levels. Using triplicate experiments, we show that measurement variations using our method are low with a mean of variation coefficients of <1%.https://www.future-science.com/doi/10.2144/05382RR05 |
| spellingShingle | Van Luu-The Nathalie Paquet Ezequiel Calvo Jean Cumps Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction BioTechniques |
| title | Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction |
| title_full | Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction |
| title_fullStr | Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction |
| title_full_unstemmed | Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction |
| title_short | Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction |
| title_sort | improved real time rt pcr method for high throughput measurements using second derivative calculation and double correction |
| url | https://www.future-science.com/doi/10.2144/05382RR05 |
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