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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

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Author(s):
Ferreira, Elisa N. [1, 2] ; Maschietto, Mariana [1] ; Silva, Sabrina D. [1] ; Brentani, Helena [3] ; Carraro, Dirce M. [1]
Total Authors: 5
Affiliation:
[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
Total Affiliations: 3
Document type: Journal article
Source: DIAGNOSTIC MOLECULAR PATHOLOGY; v. 19, n. 1, p. 45-53, MAR 2010.
Web of Science Citations: 9
Abstract

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)