RESUMO
For the preservation of tissue samples, formalin fixation followed by paraffin embedding (FFPE) has been the method of choice for decades, mainly because it maintains the morphologic characteristics of the original tissue particularly preserved, as well as its genetic material. FFPE cells can be used to perform molecular tests, such as conventional (c) or quantitative (q) reverse transcriptase polymerase chain reaction (RT-PCR), in retrospective investigations. However, extracting RNA from archived FFPE tissues is a challenging procedure, as it requires time and the use of complex extraction methods. As specific FFPE extraction methods are not always available in the laboratories, the objective of this study was to evaluate the performance of a method based on phenol-chloroform (PC) and 2 commercial methods for RNA extraction, adapting their protocols for FFPE tissues. For this study, a pool of FFPE tissues underwent RNA extraction by PC, QIAmp Viral RNA Mini, and RNeasy Mini Kit. Both the RT-cPCR and the RT-qPCR results were favorable, demonstrating the viability of the RNA. As these results expanded the alternatives for low-budget FFPE extraction, the choice of the ideal method to be used will depend on the availability of reagents and kits.
Assuntos
Inclusão em Parafina/métodos , RNA/isolamento & purificação , Fixação de Tecidos , Encéfalo/metabolismo , Clorofórmio/química , Formaldeído , Humanos , Fígado/metabolismo , Pulmão/metabolismo , Fenol/química , RNA Ribossômico 18S/isolamento & purificação , RNA Ribossômico 28S/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Baço/metabolismo , Fixação de Tecidos/métodosRESUMO
The analysis of total protoscolex DNA and some rDNA recombinats of Echinococcus granulosus by restriction endonuclease mapping and hybridization to rDNA probes indicated the complex organization of the ribosomal RNA genes and that some repeat units are larger than 15 kb. The nontranscribed spacer can be up to 13 kb in length in some repeat units. Restriction site polymorphisms was detected mainly in the nontranscribed spacer regions although some polymorphisms was also observed in the 28S rRNA coding region. On the basis of Southern blot hybridization using EcoRi-digested genomic DNA, we conclude that the repeat units containing an extra EcoRI site are present almost in the same proportion as the repeat units without the extra EcoRi site in the 28S rRNA coding region