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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

Establishment of MLPA reference probe quality by statistical analyses of results obtained from cohorts comprising unaffected and affected individuals, respectively. To ensure a normal distribution of the obtained data for the reference probes AFAP1Lb (A) and AFAP1La (B) in all analyzed cohorts, the z score and Q-Q plots were established based upon data from 48 unaffected individuals and 145 breast tumors (affected individuals). The z score based histograms and Q-Q plots for AFAP1Lb followed a normal distribution for both cohorts, and the reference probe was included in the study (A). Data obtained for the reference probe AFAP1La were not normally distributed as illustrated by the z score based histograms and the Q-Q plots for both cohorts. The probe was excluded from further studies (B).
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Figure 1: Establishment of MLPA reference probe quality by statistical analyses of results obtained from cohorts comprising unaffected and affected individuals, respectively. To ensure a normal distribution of the obtained data for the reference probes AFAP1Lb (A) and AFAP1La (B) in all analyzed cohorts, the z score and Q-Q plots were established based upon data from 48 unaffected individuals and 145 breast tumors (affected individuals). The z score based histograms and Q-Q plots for AFAP1Lb followed a normal distribution for both cohorts, and the reference probe was included in the study (A). Data obtained for the reference probe AFAP1La were not normally distributed as illustrated by the z score based histograms and the Q-Q plots for both cohorts. The probe was excluded from further studies (B).

Mentions: Peak heights of the reference probes were analyzed in a Gaussian Q-Q plot and in a histogram, where all reference probe peaks have to show the same normalization pattern in both affected and unaffected individuals; otherwise, the probe is omitted from further studies 1. If the samples were overloaded, dilution of the MLPA product should be avoided as it may affect the final result. Instead, we suggest changing the injection time or voltage according to recommendations from MRC Holland. If the problem persists, the volume of probes in the probe mix can be decreased as well as the MLPA product before the capillary electrophoresis. The important issue is to treat all samples from unaffected and affected individuals in exactly the same way throughout the MLPA analysis.


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)

Establishment of MLPA reference probe quality by statistical analyses of results obtained from cohorts comprising unaffected and affected individuals, respectively. To ensure a normal distribution of the obtained data for the reference probes AFAP1Lb (A) and AFAP1La (B) in all analyzed cohorts, the z score and Q-Q plots were established based upon data from 48 unaffected individuals and 145 breast tumors (affected individuals). The z score based histograms and Q-Q plots for AFAP1Lb followed a normal distribution for both cohorts, and the reference probe was included in the study (A). Data obtained for the reference probe AFAP1La were not normally distributed as illustrated by the z score based histograms and the Q-Q plots for both cohorts. The probe was excluded from further studies (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Establishment of MLPA reference probe quality by statistical analyses of results obtained from cohorts comprising unaffected and affected individuals, respectively. To ensure a normal distribution of the obtained data for the reference probes AFAP1Lb (A) and AFAP1La (B) in all analyzed cohorts, the z score and Q-Q plots were established based upon data from 48 unaffected individuals and 145 breast tumors (affected individuals). The z score based histograms and Q-Q plots for AFAP1Lb followed a normal distribution for both cohorts, and the reference probe was included in the study (A). Data obtained for the reference probe AFAP1La were not normally distributed as illustrated by the z score based histograms and the Q-Q plots for both cohorts. The probe was excluded from further studies (B).
Mentions: Peak heights of the reference probes were analyzed in a Gaussian Q-Q plot and in a histogram, where all reference probe peaks have to show the same normalization pattern in both affected and unaffected individuals; otherwise, the probe is omitted from further studies 1. If the samples were overloaded, dilution of the MLPA product should be avoided as it may affect the final result. Instead, we suggest changing the injection time or voltage according to recommendations from MRC Holland. If the problem persists, the volume of probes in the probe mix can be decreased as well as the MLPA product before the capillary electrophoresis. The important issue is to treat all samples from unaffected and affected individuals in exactly the same way throughout the MLPA analysis.

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