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J Biomol Tech. 2010 September; 21(3 Suppl): S43.
PMCID: PMC2918069

New Technologies for FFPE Samples Improved RNA Isolation and Novel cDNA Priming for qPCR and for Universal mRNA Amplification

R. Jaggi2 and S. Quabius3
1AmpTec GmbH, Hamburg, Germany;
2Department of Clinical Research, University of Bern, Switzerland;
3Institute for Immunology, UKSH, Kiel, Germany

Abstract

RP-72

Archival FFPE (formalin-fixed paraffin-embedded) samples have been collected over decades in routine clinical procedures and they harbour a great wealth of information, including RNA, Although the RNA is severely degraded and poses additional challenges due to inter- and intramolecular cross-linking and base modifications, mining of gene expression data is still possible and extracted information about differential gene expression is comparable to data from fresh-frozen samples, even on a quantitative level. The combination of a novel procedure for RNA liberation and demodification results in highly reproducible data in RT-qPCR studies [1] and can be used to determine RNA profiles of cancer samples [2]. Our novel TR priming strategy combines the advantages of oligo-dT (priming near the 3'end and selection against rRNAs) with random priming (works with mRNA fragments without poly-A: no requirement for a universal target sequence). This TR priming can be used for cDNA synthesis with FFPE RNA templates, followed by qPCR analyses or combined with mRNA amplification and labelling for genome-wide microarray gene expression profiling. Data will be presented to demonstrate the advanced recovery of FFPE RNAs: quality control with RNA profiles (Agilent Bioanalyzer), comparison of expression profiles obtained with RNAs from fresh-frozen and from FFPE samples, as determined by RT-qPCR as well as Affymetrix microarray analysis. [1] Oberli et al. (2008) Expression profiling with RNA from formalin-fixed, paraffin-embedded material. BMC Medical Genomics 2008, 1:9. [2] Schobesberger et al. (2008) Gene expression variation between distinct areas of breast cancer measured from paraffin-embedded tissue cores. BMC Cancer 2008, 8:343.


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