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Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.

Whale AS, Huggett JF, Cowen S, Speirs V, Shaw J, Ellison S, Foy CA, Scott DJ - Nucleic Acids Res. (2012)

Bottom Line: One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements.An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma.Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration.

View Article: PubMed Central - PubMed

Affiliation: LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.

ABSTRACT
One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.

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Comparison of HER2:RNase P ratio in breast cancer cell line genomic DNA using digital and quantitative real-time PCR. (a) Software-generated heat map showing a single panel in a 48.770 dPCR array that contains 770 chambers with positive (white) and negative (black) amplification signals. One representative dPCR panel is shown for each gDNA sample and assay with the number of positive chambers shown in the top right corner of the panel. Positive and negative chambers were used to calculate the number of molecules per panel and the HER2:RNase P ratio for the gDNA sample. The NTC panels for both assays had no positive chambers. (b) qPCR (n = 8 wells) and dPCR (n = 4 panels) gave similar HER2:RNase P ratios for all BC gDNA except the SK-BR-3 gDNA that was significantly higher by qPCR compared with dPCR (asterisk). Data is presented on a log scale and error bars represent 95% CIs.
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gks203-F1: Comparison of HER2:RNase P ratio in breast cancer cell line genomic DNA using digital and quantitative real-time PCR. (a) Software-generated heat map showing a single panel in a 48.770 dPCR array that contains 770 chambers with positive (white) and negative (black) amplification signals. One representative dPCR panel is shown for each gDNA sample and assay with the number of positive chambers shown in the top right corner of the panel. Positive and negative chambers were used to calculate the number of molecules per panel and the HER2:RNase P ratio for the gDNA sample. The NTC panels for both assays had no positive chambers. (b) qPCR (n = 8 wells) and dPCR (n = 4 panels) gave similar HER2:RNase P ratios for all BC gDNA except the SK-BR-3 gDNA that was significantly higher by qPCR compared with dPCR (asterisk). Data is presented on a log scale and error bars represent 95% CIs.

Mentions: In order to investigate the accuracy of dPCR for CNV measurement, we used three BC cell lines with different HER2 gene copy number as a model of gene amplification. The RNase P assay was used as the reference gene for the diploid control. Assay optimization was performed using qPCR for HER2 and RNase P assays (Supplementary Figure S1). For dPCR, absolute quantification of HER2 and RNase P molecules were calculated from the number of positive counts per panel based on the Poisson distribution for the number of molecules in each chamber (Figure 1a).Figure 1.


Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.

Whale AS, Huggett JF, Cowen S, Speirs V, Shaw J, Ellison S, Foy CA, Scott DJ - Nucleic Acids Res. (2012)

Comparison of HER2:RNase P ratio in breast cancer cell line genomic DNA using digital and quantitative real-time PCR. (a) Software-generated heat map showing a single panel in a 48.770 dPCR array that contains 770 chambers with positive (white) and negative (black) amplification signals. One representative dPCR panel is shown for each gDNA sample and assay with the number of positive chambers shown in the top right corner of the panel. Positive and negative chambers were used to calculate the number of molecules per panel and the HER2:RNase P ratio for the gDNA sample. The NTC panels for both assays had no positive chambers. (b) qPCR (n = 8 wells) and dPCR (n = 4 panels) gave similar HER2:RNase P ratios for all BC gDNA except the SK-BR-3 gDNA that was significantly higher by qPCR compared with dPCR (asterisk). Data is presented on a log scale and error bars represent 95% CIs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367212&req=5

gks203-F1: Comparison of HER2:RNase P ratio in breast cancer cell line genomic DNA using digital and quantitative real-time PCR. (a) Software-generated heat map showing a single panel in a 48.770 dPCR array that contains 770 chambers with positive (white) and negative (black) amplification signals. One representative dPCR panel is shown for each gDNA sample and assay with the number of positive chambers shown in the top right corner of the panel. Positive and negative chambers were used to calculate the number of molecules per panel and the HER2:RNase P ratio for the gDNA sample. The NTC panels for both assays had no positive chambers. (b) qPCR (n = 8 wells) and dPCR (n = 4 panels) gave similar HER2:RNase P ratios for all BC gDNA except the SK-BR-3 gDNA that was significantly higher by qPCR compared with dPCR (asterisk). Data is presented on a log scale and error bars represent 95% CIs.
Mentions: In order to investigate the accuracy of dPCR for CNV measurement, we used three BC cell lines with different HER2 gene copy number as a model of gene amplification. The RNase P assay was used as the reference gene for the diploid control. Assay optimization was performed using qPCR for HER2 and RNase P assays (Supplementary Figure S1). For dPCR, absolute quantification of HER2 and RNase P molecules were calculated from the number of positive counts per panel based on the Poisson distribution for the number of molecules in each chamber (Figure 1a).Figure 1.

Bottom Line: One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements.An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma.Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration.

View Article: PubMed Central - PubMed

Affiliation: LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.

ABSTRACT
One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.

Show MeSH
Related in: MedlinePlus