Nucleic acid sequence-based amplification in formalin-fixed and paraffin-embedded breast-cancer tissues.
OBJECTIVE
To evaluate the nucleic acid sequence-based amplification (NASBA) technique to amplify mRNA isolated from formalin-fixed and paraffin-embedded (FFPE) breast-cancer tissues.


METHODS
RNA was extracted from archived, 10-year-old FFPE tissues, and selected genes, namely ribosomal protein S18 (RPS18), epidermal growth factor receptor 2 (HER2), estrogen receptor alpha (ERα), Y box binding protein (YBX-1), matrix metallopeptidase 11 (MMP11), caspase 8 (CASP8) and superoxide dismutase 2 (SOD2), were amplified by NASBA.


RESULTS
Despite strong degradation of the template, RNA amplification of all tested genes resulted in strong hybridisation signals. Sensitivity tests showed that the RPS18 NASBA assay was more sensitive than real-time RT-PCR used as a reference method. The sensitivity of the HER2, ERα, MMP11, YBX1, CASP8 and SOD2 NASBA assay was comparable with RT-PCR targeted to the respective genes.


CONCLUSIONS
The results obtained indicate that NASBA is suitable to amplify with high specificity and sensitivity, even strongly degraded RNA isolated from FFPE tissues, and therefore can complement already-existing amplification techniques such as RT-PCR for analysis of such tissues.
Department of Gynecology and Obstetrics, University Hospital Freiburg, Freiburg, Germany.
Ulrike
U
Riehle
Andreas
A
Mader
Thomas
T
Brandstetter
Jürgen
J
Rühe
Axel
A
zur Hausen
Elmar
E
Stickeler
D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D005240:Feasibility Studies; D005260:Female; D005557:Formaldehyde; D052138:Genes, Neoplasm; D018734:Genes, erbB-2; D006801:Humans; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods
D012333:RNA, Messenger; D012334:RNA, Neoplasm; D005557:Formaldehyde