Quantitative expression profiling of highly degraded RNA from formalin-fixed, paraffin-embedded breast tumor biopsies by oligonucleotide microarrays.
Microarray-based gene expression profiling is well suited for parallel quantitative analysis of large numbers of RNAs, but its application to cancer biopsies, particularly formalin-fixed, paraffin-embedded (FFPE) archived tissues, is limited by the poor quality of the RNA recovered. This represents a serious drawback, as FFPE tumor tissue banks are available with clinical and prognostic annotations, which could be exploited for molecular profiling studies, provided that reliable analytical technologies are found. We applied and evaluated here a microarray-based cDNA-mediated annealing, selection, extension and ligation (DASL) assay for analysis of 502 mRNAs in highly degraded total RNA extracted from cultured cells or FFPE breast cancer (MT) biopsies. The study included quantitative and qualitative comparison of data obtained by analysis of the same RNAs with genome-wide oligonucleotide microarrays vs DASL arrays and, by DASL, before and after extensive in vitro RNA fragmentation. The DASL-based expression profiling assay applied to RNA extracted from MCF-7 cells, before or after 24 h stimulation with a mitogenic dose of 17beta-estradiol, consistently allowed to detect hormone-induced gene expression changes following extensive RNA degradation in vitro. Comparable results where obtained with tumor RNA extracted from FFPE MT biopsies (6 to 19 years old). The method proved itself sensitive, reproducible and accurate, when compared to results obtained by microarray analysis of RNA extracted from snap-frozen tissue of the same tumor.
Ravo
Maria
M
Dipartimento di Patologia generale and Centro Grandi Apparecchiature, Seconda Università degli Studi di Napoli, Napoli, Italy.
Mutarelli
Margherita
M
Ferraro
Lorenzo
L
Grober
Olì Maria Victoria
OM
Paris
Ornella
O
Tarallo
Roberta
R
Vigilante
Alessandra
A
Cimino
Daniela
D
De Bortoli
Michele
M
Nola
Ernesto
E
Cicatiello
Luigi
L
Weisz
Alessandro
A
D001706:Biopsy; D001943:Breast Neoplasms / Q000473:pathology*; D018270:Carcinoma, Ductal, Breast / Q000473:pathology*; D045744:Cell Line, Tumor; D005260:Female; D005557:Formaldehyde; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding; D012334:RNA, Neoplasm / Q000032:analysis*; D015203:Reproducibility of Results
D012334:RNA, Neoplasm; D005557:Formaldehyde