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

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.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.Copy number variations (CNV), including deletions or duplications of whole chromosomes and minor fragments down to single exons are implicated in the onset and risk of human congenital and somatic diseases [1]. The Multiplex Ligation-dependent Probe Amplification (MLPA) is widely used both in research and clinical laboratories and is a promising methodology to assess CNV [2]. MLPA assays provide highly detailed information on CNV, from chromosome aneuploidy down to single exons. The methodology is fast and straightforward to perform, standard laboratory equipment is used, and a limited amount of DNA (20 ng/analysis) is necessary to provide high quality and reproducible results. Ligation-dependent PCR amplification was first described by Hsuih et al. [3] further developed by Schouten et al. [4] and a growing number of predesigned gene specific MLPA assays is commercially available from MRC Holland http://www.mrc-holland.com webcite. The MLPA methodology is versatile and can be applied to different analys