Evaluation of quantitative rt-PCR using nonamplified and amplified RNA.
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.
Ferreira
Elisa N
EN
Laboratory of Genomics and Molecular Biology-A.C. Camargo Hospital, Institute of Biosciences, University of São Paulo (USP), São Paulo, Brazil.
Maschietto
Mariana
M
Silva
Sabrina D
SD
Brentani
Helena
H
Carraro
Dirce M
DM
D000199:Actins / Q000235:genetics; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D057089:Pathology, Molecular / Q000379:methods* / Q000592:standards; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards
D000199:Actins; D012313:RNA