Multicentre validation study of nucleic acids extraction from FFPE tissues.
In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.
Bonin
Serena
S
ACADEM Department-University of Trieste and ICGEB, University of Trieste, Cattinara Hospital, 34149 Trieste, Italy.
Hlubek
Falk
F
Benhattar
Jean
J
Denkert
Carsten
C
Dietel
Manfred
M
Fernandez
Pedro L
PL
Höfler
Gerald
G
Kothmaier
Hannelore
H
Kruslin
Bozo
B
Mazzanti
Chiara Maria
CM
Perren
Aurel
A
Popper
Helmuth
H
Scarpa
Aldo
A
Soares
Paula
P
Stanta
Giorgio
G
Groenen
Patricia J T A
PJ
D005557:Formaldehyde; D006801:Humans; D009696:Nucleic Acids / Q000032:analysis / Q000302:isolation & purification*; D016612:Paraffin Embedding; D057089:Pathology, Molecular / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation
D009696:Nucleic Acids; D005557:Formaldehyde