Multiplex loss of heterozygosity analysis by using single or very few cells

Abstract

Loss of heterozygosity (LOH) is an indication of tumor suppressor gene inactivation. However, loss of heterozygosity analysis has been limited to either a small scale or to very few genetic markers. To significantly increase the scale of study and to include a large number of markers in the analysis, experimental conditions were established for using single cells or single cell equivalent with 10 markers typed simultaneously. Under these conditions, the allele amplification failure rate was 3.7% when single tissue cultured human cells were used. When 30 cells from a 5- micro m paraffin-archived breast tumor tissue section were used, the failure rates were 0% for four of the five heterozygous loci and 10% for the fifth. Small amplification failure rates (6.1% and 6.7% on average) were observed when 5 or 10 cells from paraffin-archived breast tissue were used. These results indicate that with polymerase chain reaction (PCR) primers of high quality, it is possible to obtain reliable results by using single cells from fresh tissue or very few cells from paraffin-archived specimens. The results also show the importance of including replicates, using primers of high quality, and optimizing PCR conditions when a limited amount of material is used for the assay. The feasibility of LOH analysis with very few paraffin-embedded breast cancer tissues was demonstrated.

Figure 1.

Schematic presentation of the multiplex PCR protocol. Three loci are shown.

Figure 2.

Part of results from LOH analysis with single or very few cells. M: MspI digest of pBR322 DNA as molecular markers. As shown, the “+” allele of locus 2 is missing in lanes 3 and 9 (lanes are counted from left to right), and the “−” allele is missing in lane 12. The “−” allele of locus 10 is missing in lane 6.

Figure 3.

Part of the results from LOH analysis with 30 breast cancer cells. Eight samples with either complete LOH or allelic reduction for the GLANS (−) allele are shown on the left and results from genotyping with normal components from the same tissue specimen are shown on the right. A non-specific band that does not affect detection is indicated by the arrow.