Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results
Abstract Background Real time PCR (rtPCR) is a quantitative assay to determine the relative DNA copy number in a sample versus a reference. The $$\Delta C_T$$ Δ C T method is the standard for the analysis of the output data generated by an rtPCR experiment. We developed an alternative based on fitti...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2017-11-01
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| Series: | BMC Research Notes |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13104-017-2913-1 |
| _version_ | 1818083180748472320 |
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| author | Paulo Refinetti David Warren Stephan Morgenthaler Per O. Ekstrøm |
| author_facet | Paulo Refinetti David Warren Stephan Morgenthaler Per O. Ekstrøm |
| author_sort | Paulo Refinetti |
| collection | DOAJ |
| description | Abstract Background Real time PCR (rtPCR) is a quantitative assay to determine the relative DNA copy number in a sample versus a reference. The $$\Delta C_T$$ Δ C T method is the standard for the analysis of the output data generated by an rtPCR experiment. We developed an alternative based on fitting a robust regression to the rtPCR signal. This new data analysis tool reduces potential biases and does not require all of the compared DNA fragments to have the same PCR efficiency. Results Comparing the two methods when analysing 96 identical PCR preparations showed similar distributions of the estimated copy numbers. Estimating the efficiency with the $$\Delta C_T$$ Δ C T method, however, required a dilution series, which is not necessary for the robust regression method. We used rtPCR to quantify mitochondrial DNA (mtDNA) copy numbers in three different tissues types: breast, colon and prostate. For each type, normal tissue and a tumor from the same three patients were analysed. This gives a total of six samples. The mitochondrial copy number is estimated to lie between 200 and 300 copies per cell. Similar results are obtained when using the robust regression or the $$\Delta C_T$$ Δ C T method. Confidence ratios were slightly narrower for the robust regression. The new data analysis method has been implemented as an R package. |
| first_indexed | 2024-12-10T19:33:54Z |
| format | Article |
| id | doaj.art-82fdcb7623a647d6849422dbb0674193 |
| institution | Directory Open Access Journal |
| issn | 1756-0500 |
| language | English |
| last_indexed | 2024-12-10T19:33:54Z |
| publishDate | 2017-11-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Research Notes |
| spelling | doaj.art-82fdcb7623a647d6849422dbb06741932022-12-22T01:36:10ZengBMCBMC Research Notes1756-05002017-11-011011710.1186/s13104-017-2913-1Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR resultsPaulo Refinetti0David Warren1Stephan Morgenthaler2Per O. Ekstrøm3Ecole Polytechnique Féderale de LausanneDepartment of Medical Biochemistry, RadiumhospitalEcole Polytechnique Féderale de LausanneDepartment of Tumor Biology, RadiumhospitalAbstract Background Real time PCR (rtPCR) is a quantitative assay to determine the relative DNA copy number in a sample versus a reference. The $$\Delta C_T$$ Δ C T method is the standard for the analysis of the output data generated by an rtPCR experiment. We developed an alternative based on fitting a robust regression to the rtPCR signal. This new data analysis tool reduces potential biases and does not require all of the compared DNA fragments to have the same PCR efficiency. Results Comparing the two methods when analysing 96 identical PCR preparations showed similar distributions of the estimated copy numbers. Estimating the efficiency with the $$\Delta C_T$$ Δ C T method, however, required a dilution series, which is not necessary for the robust regression method. We used rtPCR to quantify mitochondrial DNA (mtDNA) copy numbers in three different tissues types: breast, colon and prostate. For each type, normal tissue and a tumor from the same three patients were analysed. This gives a total of six samples. The mitochondrial copy number is estimated to lie between 200 and 300 copies per cell. Similar results are obtained when using the robust regression or the $$\Delta C_T$$ Δ C T method. Confidence ratios were slightly narrower for the robust regression. The new data analysis method has been implemented as an R package.http://link.springer.com/article/10.1186/s13104-017-2913-1rtPCRRobust regressionMitochondrial DNA |
| spellingShingle | Paulo Refinetti David Warren Stephan Morgenthaler Per O. Ekstrøm Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results BMC Research Notes rtPCR Robust regression Mitochondrial DNA |
| title | Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results |
| title_full | Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results |
| title_fullStr | Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results |
| title_full_unstemmed | Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results |
| title_short | Quantifying mitochondrial DNA copy number using robust regression to interpret real time PCR results |
| title_sort | quantifying mitochondrial dna copy number using robust regression to interpret real time pcr results |
| topic | rtPCR Robust regression Mitochondrial DNA |
| url | http://link.springer.com/article/10.1186/s13104-017-2913-1 |
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