title: Comparison of two methods of RNA extraction from formalin-fixed paraffin-embedded tissue specimens.
abstract: The present study aimed to compare two different methods of extracting RNA from formalin-fixed paraffin-embedded (FFPE) specimens of diffuse large B-cell lymphoma (DLBCL). We further aimed to identify possible influences of variables--such as tissue size, duration of paraffin block storage, fixative type, primers used for cDNA synthesis, and endogenous genes tested--on the success of amplification from the samples. Both tested protocols used the same commercial kit for RNA extraction (the RecoverAll Total Nucleic Acid Isolation Optimized for FFPE Samples from Ambion). However, the second protocol included an additional step of washing with saline buffer just after sample rehydration. Following each protocol, we compared the RNA amount and purity and the amplification success as evaluated by standard PCR and real-time PCR. The results revealed that the extra washing step added to the RNA extraction process resulted in significantly improved RNA quantity and quality and improved success of amplification from paraffin-embedded specimens.
authors.affiliation: University of São Paulo's Medical School (FMUSP), 05403-000 São Paulo, SP, Brazil ; Hospital das Clínicas da FMUSP, Laboratório de Imunopatologia, Avenida Doutor Enéas Carvalho de Aguiar, 155/1° Andar, Cerqueira César, 05403-000 São Paulo, SP, Brazil.
authors.forename: Gisele Rodrigues
authors.initials: GR
authors.lastname: Gouveia
authors.affiliation: FMUSP, São Paulo's Blood Center/Fundação Pró-Sangue's, Molecular Biology Department, 05403-000 São Paulo, SP, Brazil.
authors.forename: Suzete Cleusa
authors.initials: SC
authors.lastname: Ferreira
authors.affiliation: University of São Paulo's Medical School (FMUSP), 05403-000 São Paulo, SP, Brazil.
authors.forename: Jerenice Esdras
authors.initials: JE
authors.lastname: Ferreira
authors.affiliation: FMUSP's Hospital das Clínicas (HC) Pathology Service, 05403-000 São Paulo, SP, Brazil.
authors.forename: Sheila Aparecida Coelho
authors.initials: SA
authors.lastname: Siqueira
authors.affiliation: FMUSP, State of São Paulo Cancer Institute (ICESP), HC/FMUSP Central Institute (IC) Hematology Service, 05403-000 São Paulo, SP, Brazil.
authors.forename: Juliana
authors.initials: J
authors.lastname: Pereira
mesh_terms: D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D016612:Paraffin Embedding; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000737:chemistry / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
chemical_list: D005404:Fixatives; D012334:RNA, Neoplasm; D005557:Formaldehyde; D012313:RNA
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