BACKGROUND: The comparison of molecular genetic changes in healthy and pathological tissues has historically led to the identification of oncogenes and tumor suppressor genes. It is very common that studies investigating loss of heterozygosity are carried out retrospectively on paraffin-embedded samples. METHODS: In this study, we evaluated the power of pyrosequencing for determining the loss of heterozygotic regions. The present method uses the fact that pyrosequencing is an accurate, sensitive and reproducible technique. The method is also simple to perform, with results available in 96-well format, making the assays amenable to automation. Thus, we analyzed nine single nucleotide polymorphisms along 1 Mb between the EMSY and PAK1 genes on 11q13, a region frequently rearranged in different tumors and cell lines. We assessed the study using samples from breast cancer and thyroid cancer biopsies. RESULTS AND CONCLUSIONS: We conclude that this technique is capable of detecting variations of >10% in allele loss. However, strong allele imbalances were detected, depending on the origin of the sample. Seven out of the nine markers used exhibited differential allele amplification, depending on the DNA quality (p<0.01).
BACKGROUND: The comparison of molecular genetic changes in healthy and pathological tissues has historically led to the identification of oncogenes and tumor suppressor genes. It is very common that studies investigating loss of heterozygosity are carried out retrospectively on paraffin-embedded samples. METHODS: In this study, we evaluated the power of pyrosequencing for determining the loss of heterozygotic regions. The present method uses the fact that pyrosequencing is an accurate, sensitive and reproducible technique. The method is also simple to perform, with results available in 96-well format, making the assays amenable to automation. Thus, we analyzed nine single nucleotide polymorphisms along 1 Mb between the EMSY and PAK1 genes on 11q13, a region frequently rearranged in different tumors and cell lines. We assessed the study using samples from breast cancer and thyroid cancer biopsies. RESULTS AND CONCLUSIONS: We conclude that this technique is capable of detecting variations of >10% in allele loss. However, strong allele imbalances were detected, depending on the origin of the sample. Seven out of the nine markers used exhibited differential allele amplification, depending on the DNA quality (p<0.01).
Authors: M Hidalgo; M E Saez; F J Martinez-Tello; F J Moron; E Ferrero-Herrero; M Labalde-Martinez; D Rigopoulou; C Ballestin-Carcavilla; A Ruiz; J L Royo; R Ramirez-Lorca Journal: J Endocrinol Invest Date: 2008-07 Impact factor: 4.256
Authors: Manuel Hidalgo; Jose Jorge Galan; Carmen Sáez; Eduardo Ferrero; Carolina Castilla; Reposo Ramirez-Lorca; Pablo Pelaez; Agustin Ruiz; Miguel A Japón; Jose Luis Royo Journal: BMC Cancer Date: 2008-04-21 Impact factor: 4.430