Comparison of allelic ratios from paired blood and paraffin-embedded normal tissue for use in a polymerase chain reaction to assess loss of heterozygosity

Background: One method to assess loss of heterozygosity (LOH) of various ge nes is the amplification of DNA from neoplastic tissue by using microsatell ite markers. LOH can best be considered on a quantitative basis as a compar ison of allelic ratios of neoplastic tissue to that of the normal control. We will illustrate through quantitative methods the importance of using the appropriate controls when determining allelic loss. Methods and Results: DNA extracted from 28 paired blood and formalin-fu;ed, paraffin-embedded normal mucosal tissue was amplified using the DP1 micros atellite marker, consisting of a variable number of CA repeats. This marker is located within the D5S346 (DP1) region on chromosome 5 and is linked to the adenomatous polyposis coli gene. Allelic ratios were calculated after scanning autoradiographs on a densitometer. Ratio values approaching 1 were observed when the two alleles were close in molecular weight, whereas rati os less than 1 were detected when the two alleles had very different molecu lar weights. This discrepancy was more pronounced in paraffin-embedded tiss ue than with blood samples. Conclusion: For LOH amplification assays, it is best to use normal control samples that are of the same tissue source as the neoplastic sample being a nalyzed. When assessing LOH in neoplastic tissue, a quantitative value rath er than visual assessment of the alleles should be considered. The values m ay be normalized by dividing the ratio of the two tumor alleles by the rati o of the two normal alleles.