RESUMO
Relative gene quantification by quantitative reverse transcription PCR (qRT-PCR) is an accurate technique only when a correct normalization strategy is carried out. Some of the most commonly genes used as reference have demonstrated variation after interferon (IFN) treatments. In this work we evaluated the suitability of seven reference genes (RGs) [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase (HMBS), ß-2Microglobulin (B2M), ribosomal RNA subunits 18S and 28S, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) and the RNA helicase (DDX5)] for use in qRT-PCR assays in the glioblastoma-derived cell line U87MG treated with IFNα, IFNγ or a co-formulated combination of both IFNs (HeberPAG); untreated cell lines were included as control. Data was analyzed using geNorm and NormFinder softwares. The expression stability of the seven RGs decreased in order of DDX5/GAPDH/HMBS, 18S rRNA, YWHAZ, 28S rRNA and B2M. qRT-PCR analyses demonstrated that DDX5, GAPDH and HMBS were among the best stably expressed markers under all conditions. Both, geNorm and NormFinder, analyses proposed same RGs as the least variables. Evaluation of the expression levels of two target genes utilizing different endogenous controls, using REST-MCS software, revealed that the normalization method applied might introduce errors in the estimation of relative quantities. We concluded that when qRT-PCR is designed for studies of gene expression in U87MG cell lines treated with IFNs type I and II or their combinations, the use of all three GAPDH, HMBS and DDX5 (or their combinations in pairs) as RGs for data normalizations is recommended.
Assuntos
Genes Neoplásicos/genética , Interferons/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Transcrição Reversa/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Estudos de Associação Genética , Humanos , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Padrões de Referência , Transcrição Reversa/efeitos dos fármacos , SoftwareRESUMO
El grupo imidazol de la cadena lateral de la histidina ha causado problemas en la síntesis en fase sólida de péptidos. El carácter básico de este grupo hace necesario su protección para evitar reacciones colaterales durante la síntesis. En nuestro trabajo se reporta la eliminación del dinitrofenilo (DnP), grupo protector del imidazol de la histidina, bajo las condiciones de desprotección del grupo Fmoc- (20 porciento piperidina/DMF). Esta reacción colateral de eliminación del Dnp bajo las condiciones de desprotección del Fmoc- es perjudicial cuando se combinan las dos estrategias Boc/Bzl y Fmoc/t-But en la síntesis de péptidos. Para demostrar la eliminación del Dnp bajo slas condiciones de eliminación del Fmoc-, se sintetizó un pentapéptido (GHALG) en fase sólida. Se eliminó el Dnp utilizando el procedimiento convencional y por tratamiento con 20 porciento piperidina/DMF. La eliminación del Dnp con 20 porciento piperidine/DMF se demostró por la prueba de Pauly, por cromatografía líquida de alta resolución y por espectrometría de masas. El análisis de aminoácidos mostró un 84 porciento de eliminación del Dnp (AU)
