Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Autor(es)

Ferreira, Elisa N. ^{[1, 2]} ; Maschietto, Mariana ^[1] ; Silva, Sabrina D. ^[1] ; Brentani, Helena ^[3] ; Carraro, Dirce M. ^[1]

Afiliação do(s) autor(es)

^[1] AC Camargo Hosp, Lab Genom & Mol Biol, BR-01509010 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Inst Biosci, Dept Genet & Evolut Biol, Sao Paulo - Brazil

^[3] AC Camargo Hosp, Biotechnol Lab, BR-01509010 Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: DIAGNOSTIC MOLECULAR PATHOLOGY; v. 19, n. 1, p. 45-53, MAR 2010.
• Citações Web of Science: 9

Resumo

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data. (AU)

• Processo FAPESP: 05/56289-2 - Abordagens para identificação de variantes de splicing associadas ao câncer de mama sob influência da alta expressão do oncogene ERBB2
• Beneficiário: Elisa Napolitano e Ferreira
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 06/00081-7 - Caracterizacao da expressao dos genes da via de sinalizacao wnt e do processo de transicao epitelio-mesenquima durante a embriogenese de rim e figado e suas implicacoes em tumores embrionarios.
• Beneficiário: Mariana Camargo Maschietto
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 06/61040-6 - Identificacao de marcadores moleculares de progressao e prognostico em carcinomas epidermoides de boca.
• Beneficiário: Sabrina Daniela da Silva
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado