Reliability and Reproducibility of a RNA Preamplification... : Diagnostic Molecular Pathology (original) (raw)

Original Articles

Reliability and Reproducibility of a RNA Preamplification Method for Low-density Array Analysis From Formalin-fixed Paraffin-embedded Breast Cancer Samples

Ciotti, Paola PhD* †; Garuti, Anna BcS‡; Ballestrero, Alberto MD‡; Cirmena, Gabriella BcS‡; Chiaramondia, Maurizio MD§; Baccini, Paola MD∥; Bellone, Emilia PhD* †; Mandich, Paola PhD, MD* †

Departments of *Neuroscience, Ophthalmology, and Genetics, Section of Medical Genetics

‡Internal Medicine, University of Genova

Units of †Medical Genetics

§Cytopathology

∥Pathology, Azienda Ospedaliera Universitaria San Martino of Genova, Genova, Italy

Reprints: Dr Paola Ciotti, PhD, Department of Neuroscience, Ophthalmology, and Genetics, Section of Medical Genetics, University of Genova c/o DIMI–Viale Benedetto XV, 6–16132 Genova, Italy (e-mail: [email protected]).

Paola Ciotti and Anna Garuti contributed equally to this work.

Abstract

Molecular signatures of tumors with prognostic and predictive value are now available. However, several technical problems prevent the performing of gene expression profiles in routine diagnostics. The highly sensitive fluorescence-based real-time quantitative reverse transcriptase-polymerase chain reaction procedure is able to analyze mRNA levels from formalin-fixed paraffin-embedded (FFPE) tissues, the most commonly available source of tumor samples with well-documented information. However, the time-dependent fragmentation and small amount of RNA extracted from FFPE requires a preliminary mRNA amplification step to amplify small amounts of mRNA without significantly distorting relative mRNA levels. The purpose of this study was to determine the performance of a commercial cDNA preamplification kit (TaqMan PreAmp Master Mix Kit) on RNA samples obtained from FFPE tissues, prepared, and archived for routine diagnosis. We tested the reliability and reproducibility of this procedure for performing low-density gene expression assay from FFPE tissue samples. The whole procedure reported herein is a powerful and reproducible approach for routine clinical purposes that can be performed even using poorer RNA quality samples and that allows the analysis of gene expression for 96 genes in stored samples.