An improved internal mammary irradiation technique in radiation treatment of locally advanced breast cancers

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135635/1/acm20084.pd

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Findings</p> <p>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 (<it>RGS8</it>), which we previously have identified as being affected by allelic imbalance by LOH analysis across <it>RGS8 </it>in the cohort comprising 145 breast tumors. Agreement between the LOH results and the results obtained by each of the three MLPA probes in <it>RGS8 </it>was found for 64%, 73%, and 91%, of the analyzed samples, respectively.</p> <p>Conclusion</p> <p>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.</p