KRAS mutation analysis in genomic DNA isolated from formalin-fixed paraffin-embedded ovarian tissue: evaluation of a strip-based reverse-hybridisation assay.
OBJECTIVE
To evaluate a reverse-hybridisation assay (strip assay) designed for the sensitive detection of 10 mutations in codons 12 and 13 of the KRAS gene. The strip assay relies on mutant-enriched PCR followed by reverse-hybridisation of biotinylated amplification products to oligonucleotide probes immobilised as an array of parallel lines on nitrocellulose test strips.


METHODS
The strip assay was used to analyse genomic DNA isolated from 120 formalin-fixed paraffin-embedded (FFPE) ovarian tissue samples. The samples were analysed in parallel using a biochip-based protocol (biochip assay) covering the same mutation spectrum, and results were compared with respect to sensitivity, specificity and operational input.


RESULTS
The strip assay identified 19 (16%) of 120 FFPE samples to carry a KRAS mutation; results were in agreement with those obtained by biochip hybridisation. Both assays had an analytical sensitivity of 1% when performed on FFPE-extracted DNA with approximately the same operational input needed for post-PCR processing. In contrast to the biochip assay, strip assay hybridisation may be automated to a large extent.


CONCLUSIONS
The strip assay is an accurate and sensitive tool for the low to medium throughput detection of KRAS mutation in genomic DNA isolated from FFPE tissue.
ViennaLab Diagnostics GmbH, Gaudenzdorfer Gürtel 43-45,Vienna, Austria. kriegshauser@viennalab.com
Gernot
G
Kriegshäuser
Veronika
V
Auner
Eva
E
Schuster
Barbara
B
Holzer
Christian
C
Oberkanins
Reinhard
R
Horvat
Paul
P
Speiser
Robert
R
Zeillinger
D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D005557:Formaldehyde; D006801:Humans; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D040081:Protein Array Analysis / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D011934:Reagent Strips; D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000379:methods; D018631:ras Proteins / Q000235:genetics*
D004273:DNA, Neoplasm; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D011934:Reagent Strips; D005557:Formaldehyde; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins