Alternative PCR protocol using a single primer set for assessing DNA quality in several tissues from a large variety of mammalian species living in areas endemic for leishmaniasis

The aim of this work was to establish a modified pre-diagnostic polymerase chain reaction (PCR) protocol using a single primer set that enables successful amplification of a highly conserved mammalian sequence in order to determine overall sample DNA quality for multiple mammalian species that inhabit areas endemic for leishmaniasis. The gene encoding interphotoreceptor retinoid-binding protein (IRBP), but not other conserved genes, was efficiently amplified in DNA samples from tail skin, ear skin, bone marrow, liver and spleen from all of the species tested. In tissue samples that were PCR-positive for Leishmania, we found that DNA from 100 percent, 55 percent and 22 percent of the samples tested resulted in a positive PCR reaction for the IRBP, beta-actin and beta-globin genes, respectively. Nucleotide sequencing of an IRBP amplicon resolved any questions regarding the taxonomical classification of a rodent, which was previously based simply on the morphological features of the animal. Therefore, PCR amplification and analysis of the IRBP amplicon are suitable for pre-diagnostically assessing DNA quality and identifying mammalian species living in areas endemic to leishmaniasis and other diseases.

Estatus da metilação da caderina-E em tecido tireóideo normal, bócio e câncer papilar de tireóide / E-Cadherin methylation status in normal thyroid tissue, goiter and papillary thyroid cancer

A descoberta de marcadores moleculares que possam predizer transformação maligna em lesões tireoidianas é de fundamental importância na prática clínica. A inativação da caderina-E é um evento inicial observado em muitas lesões epiteliais cancerígenas. Objetivo: Verificar o possível papel da metilação do gene da caderina-E na transformação maligna do tecido tireóideo. Método: A metilação do gene da caderina-E foi determinado por PCR-MS após a extração do DNA usando a técnica com bissulfato de sódio. As linhagens celulares leucêmicas MV411 e BV173 foram usadas como controles positivos. Dez amostras do sangue periférico, tecido tireóideo normal e carcinoma papilar de tireóide foram testados. Resultados: Células MV411 e BV173 mostraram apenas padrão metilado. Todas as amostras de sangue periférico, tecido tireóideo normal e carcinoma papilar de tireóide mostraram apenas padrão não metilado. Conclusão: Esses dados sugerem que a falta do padrão metilado tanto no tecido tireóideo normal como no carcinoma papilar indicam que a metilação da caderina-E não tem significância na carcinogênese dos tumores papilares de tireóide

The discovery of a molecular marker able to predict malignant transformation of thyroid lesions would be of up most importance in clinical practice. E-cadherin inactivation is an early event observed in many cancerous epithelial lesions. Objective: To verify the possible role of the E-cadherin gene methylation in malignant transformation of the thyroid tissue. Method: The methylation of the E-cadherin gene was assessed by MS-PCR after DNA extraction by using sodiumbisulphate technique. The leukemia-MV411 and BV173 cell lines were used as positive controls. Ten samples of peripheral blood, normal thyroid tissue, and papillary thyroid carcinomas were tested. Results: MV411 and BV173 cell lines showed only methylated pattern. All the samples of peripheral blood, normal thyroid tissue or papillary carcinomas showed only non-methylated pattern. Conclusion: These data suggest that the lack of methylated pattern for both normal thyroid tissue and papillary carcinomas indicates that methylation of the E-cadherin has no significance in thyroid papillary carcinogenesis