Microgenomics approach in formalin-fixed, decalcified, paraffin-embedded ameloblastoma tissue
Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, representing an invaluable resource for the elucidation of pathogenesis and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators.
However, the cellular heterogeneity of the tissues precludes the identification of cell-type specific gene expression profiles. Microgenomics implies the precise molecular analysis of very small, pure cell populations that have been microdissected from biopsies. Laser capture microdissection (LCM) enables the targeting and isolation of defined cells under direct microscopic visualization, while preserving biological molecules of interest.
RNA was isolated from laser capture microdissected cells from five formalin-fixed, decalcified, paraffin-embedded ameloblastoma specimens, capturing as few as 100 tumor cells. The Veritas platform was used, incorporating a UV laser that accelerates the speed at which pure cell populations can be collected.
Due to the limited quantity of RNA isolated from these specimens, it is necessary to perform an RNA amplification step prior to microarray analysis.
The quantity and purity of the resulting total RNA, as well as amplified RNA, were determined using the ND-1000 spectrophotometer and a 2100 bioanalyzer. Further, we performed qRT-PCR on B-Actin, measuring 3'/5' ratios to determine the level of degradation in our samples. The ratio of the PCR product for the 3’/5’ was close to 1.
Our results demonstrate that microgenomic technologies involving Laser Capture Microdissection can be successfully applied on formalin-fixed, paraffin-embedded tissue to obtain purified cell populations and generate cRNA of sufficient quality for high-density oligonucleotide microarray analysis.
Furthermore, RNA isolation was accomplished from decalcified tissue which is unprecedented in the literature to date.