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A simple way to evaluate self-designed probes for tumor specific Multiplex Ligation-dependent Probe Amplification (MLPA).

Pedersen K, Wiechec E, Madsen BE, Overgaard J, Hansen LL - BMC Res Notes (2010)

Bottom Line: MLPA is a versatile methodology and important tool in cancer research; it provides precise information on increased or decreased copy number at specific loci as opposed to loss of heterozygosity (LOH) studies based upon microsatellite analysis.Pre-designed MLPA kits and software are commercially available to analyze multiple exons, genes, and genomic regions.Agreement between the LOH results and the results obtained by each of the three MLPA probes in RGS8 was found for 64%, 73%, and 91%, of the analyzed samples, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Human Genetics, The Bartholin building, Wilhelm Meyers Allé 4, University of Aarhus, DK-8000 Aarhus C, Denmark. Lotte@humgen.au.dk.

ABSTRACT

Background: The Multiplex Ligation-dependent Probe Amplification (MLPA) is widely used for analysis of copy number variations (CNVs) in single or multiple loci. MLPA is a versatile methodology and important tool in cancer research; it provides precise information on increased or decreased copy number at specific loci as opposed to loss of heterozygosity (LOH) studies based upon microsatellite analysis. Pre-designed MLPA kits and software are commercially available to analyze multiple exons, genes, and genomic regions. However, an increasing demand for new gene specific assays makes it necessary to self-design new MLPA probes for which the available software may not be applicable. During evaluation of new self-designed reference probes, we encountered a number of problems, especially when applying the MLPA methodology to tumor samples.

Findings: DNA samples from 48 unaffected individuals and 145 breast cancer patients were used to evaluate 11 self-designed MLPA probes and determine the cut-off values for CNV, before applying the MLPA probes to normalize the target probes in a cohort of affected individuals. To test the calculation strategy, three probes were designed to cover regions in Regulator of G-protein Signaling 8 (RGS8), which we previously have identified as being affected by allelic imbalance by LOH analysis across RGS8 in the cohort comprising 145 breast tumors. Agreement between the LOH results and the results obtained by each of the three MLPA probes in RGS8 was found for 64%, 73%, and 91%, of the analyzed samples, respectively.

Conclusion: Here, we present a straightforward method, based upon the normalization pattern in both unaffected and affected individuals, to evaluate self-designed reference probes and to calculate CNV for the MLPA assay with specific focus on the difficulties when analyzing tumor DNA.

No MeSH data available.


Related in: MedlinePlus

Consistency and discrepancy between the RGS8 MLPA results and PCR deletion mapping of the same region.  Each column represents the rate of copy number variation based upon MLPA analysis of 11 tumor samples, in which RGS8 intragenic deletions previously have been established by LOH analysis followed by PCR mapping. For each of the three fragments 14, 31, and 32 agreements between the two methods are illustrated by the rate of deletion and amplification obtained by MLPA for individual samples. Cases with no CNV and agreement between the two methods comprise 3 (fr. 14), 1 (fr. 31) and 4 (fr. 32) samples, are not included in figure. The 95% CI from the normal distribution was used to set the cut-off value for amplification above 1.96 and for the deletion below -1.96.
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Figure 2: Consistency and discrepancy between the RGS8 MLPA results and PCR deletion mapping of the same region. Each column represents the rate of copy number variation based upon MLPA analysis of 11 tumor samples, in which RGS8 intragenic deletions previously have been established by LOH analysis followed by PCR mapping. For each of the three fragments 14, 31, and 32 agreements between the two methods are illustrated by the rate of deletion and amplification obtained by MLPA for individual samples. Cases with no CNV and agreement between the two methods comprise 3 (fr. 14), 1 (fr. 31) and 4 (fr. 32) samples, are not included in figure. The 95% CI from the normal distribution was used to set the cut-off value for amplification above 1.96 and for the deletion below -1.96.

Mentions: Three MLPA self-designed target probes were included in a MLPA study to validate the CNV calculation method. All probes were located within specific regions of RGS8, which previously has been identified as being affected by allelic imbalance by detailed LOH analysis in breast cancer samples (manuscript in preparation). Exact location of the affected regions was performed by PCR amplification of overlapping fragments throughout the affected region. None of these methods provide unambiguous CNV results. Previously analyzed reference probes AFAP1L1b and CFL2 were included to normalize the data. Eleven breast tumor samples, presenting either single or multiple allelic imbalances within the probe regions, were analyzed. MLPA analyses were conducted as described above. Based on the MLPA results, allelic imbalance of the fr.14 probe were confirmed in 64% (7/11), for the fr.31 probe in 91% (10/11), and for the fr.32 probe in 73% (8/11) (figure 2). Cases with no CNV and agreement between the two methods comprise 3 (fr. 14), 1 (fr. 31) and 4 (fr. 32) samples, are not included in figure 2.


A simple way to evaluate self-designed probes for tumor specific Multiplex Ligation-dependent Probe Amplification (MLPA).

Pedersen K, Wiechec E, Madsen BE, Overgaard J, Hansen LL - BMC Res Notes (2010)

Consistency and discrepancy between the RGS8 MLPA results and PCR deletion mapping of the same region.  Each column represents the rate of copy number variation based upon MLPA analysis of 11 tumor samples, in which RGS8 intragenic deletions previously have been established by LOH analysis followed by PCR mapping. For each of the three fragments 14, 31, and 32 agreements between the two methods are illustrated by the rate of deletion and amplification obtained by MLPA for individual samples. Cases with no CNV and agreement between the two methods comprise 3 (fr. 14), 1 (fr. 31) and 4 (fr. 32) samples, are not included in figure. The 95% CI from the normal distribution was used to set the cut-off value for amplification above 1.96 and for the deletion below -1.96.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913922&req=5

Figure 2: Consistency and discrepancy between the RGS8 MLPA results and PCR deletion mapping of the same region. Each column represents the rate of copy number variation based upon MLPA analysis of 11 tumor samples, in which RGS8 intragenic deletions previously have been established by LOH analysis followed by PCR mapping. For each of the three fragments 14, 31, and 32 agreements between the two methods are illustrated by the rate of deletion and amplification obtained by MLPA for individual samples. Cases with no CNV and agreement between the two methods comprise 3 (fr. 14), 1 (fr. 31) and 4 (fr. 32) samples, are not included in figure. The 95% CI from the normal distribution was used to set the cut-off value for amplification above 1.96 and for the deletion below -1.96.
Mentions: Three MLPA self-designed target probes were included in a MLPA study to validate the CNV calculation method. All probes were located within specific regions of RGS8, which previously has been identified as being affected by allelic imbalance by detailed LOH analysis in breast cancer samples (manuscript in preparation). Exact location of the affected regions was performed by PCR amplification of overlapping fragments throughout the affected region. None of these methods provide unambiguous CNV results. Previously analyzed reference probes AFAP1L1b and CFL2 were included to normalize the data. Eleven breast tumor samples, presenting either single or multiple allelic imbalances within the probe regions, were analyzed. MLPA analyses were conducted as described above. Based on the MLPA results, allelic imbalance of the fr.14 probe were confirmed in 64% (7/11), for the fr.31 probe in 91% (10/11), and for the fr.32 probe in 73% (8/11) (figure 2). Cases with no CNV and agreement between the two methods comprise 3 (fr. 14), 1 (fr. 31) and 4 (fr. 32) samples, are not included in figure 2.

Bottom Line: MLPA is a versatile methodology and important tool in cancer research; it provides precise information on increased or decreased copy number at specific loci as opposed to loss of heterozygosity (LOH) studies based upon microsatellite analysis.Pre-designed MLPA kits and software are commercially available to analyze multiple exons, genes, and genomic regions.Agreement between the LOH results and the results obtained by each of the three MLPA probes in RGS8 was found for 64%, 73%, and 91%, of the analyzed samples, respectively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Human Genetics, The Bartholin building, Wilhelm Meyers Allé 4, University of Aarhus, DK-8000 Aarhus C, Denmark. Lotte@humgen.au.dk.

ABSTRACT

Background: The Multiplex Ligation-dependent Probe Amplification (MLPA) is widely used for analysis of copy number variations (CNVs) in single or multiple loci. MLPA is a versatile methodology and important tool in cancer research; it provides precise information on increased or decreased copy number at specific loci as opposed to loss of heterozygosity (LOH) studies based upon microsatellite analysis. Pre-designed MLPA kits and software are commercially available to analyze multiple exons, genes, and genomic regions. However, an increasing demand for new gene specific assays makes it necessary to self-design new MLPA probes for which the available software may not be applicable. During evaluation of new self-designed reference probes, we encountered a number of problems, especially when applying the MLPA methodology to tumor samples.

Findings: DNA samples from 48 unaffected individuals and 145 breast cancer patients were used to evaluate 11 self-designed MLPA probes and determine the cut-off values for CNV, before applying the MLPA probes to normalize the target probes in a cohort of affected individuals. To test the calculation strategy, three probes were designed to cover regions in Regulator of G-protein Signaling 8 (RGS8), which we previously have identified as being affected by allelic imbalance by LOH analysis across RGS8 in the cohort comprising 145 breast tumors. Agreement between the LOH results and the results obtained by each of the three MLPA probes in RGS8 was found for 64%, 73%, and 91%, of the analyzed samples, respectively.

Conclusion: Here, we present a straightforward method, based upon the normalization pattern in both unaffected and affected individuals, to evaluate self-designed reference probes and to calculate CNV for the MLPA assay with specific focus on the difficulties when analyzing tumor DNA.

No MeSH data available.


Related in: MedlinePlus