Premium
Laser capture microdissection as a tool for deciphering the clinical relevance of molecular signatures of cancer cells
Author(s) -
Wittliff James L
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.118.1
Subject(s) - laser capture microdissection , biology , microbiology and biotechnology , gene expression , amplicon , cancer , microarray , microdissection , primer (cosmetics) , dna microarray , rna , gene , cancer cell , computational biology , genetics , polymerase chain reaction , chemistry , organic chemistry
Molecular basis of clinical behavior and therapeutic response of human carcinoma is poorly understood. To circumvent cellular heterogeneity and evaluate genomic and proteomic differences of normal and diseased cells, Laser Capture Microdissection (LCM) was employed to isolate distinct cell types non‐disruptively, allowing independent extraction of DNA, RNA or protein analyses under stringent conditions (e.g. RNase‐free & protease‐free). Using an extensive BioRepository, frozen human carcinoma biopsies with clinical follow‐up of 10–18 years were identified and pure populations of 1000–5000 cells were procured by LCM. Total RNA was processed for RT‐qPCR and amplified for microarray analyses (Agilent & Affymetrix). Spectrophotometric and BioAnalyzerTM (Agilent) analyses evaluated aRNA yield, purity and transcript length. Primer sets were created for qPCR (b–actin 200 base, 300 base & 400 base amplicons). FFPE‐extracted transcripts required amplification (ParadiseTM kit, Arcturus) and X3P chips (Affymetrix) requiring ∼200 bases on the 3′ end of aRNA. Gene expression profiles of pure populations of human carcinoma and stromal cells were discovered that correlated with cancer behavior. Significantly, several gene expression signatures were highly associated with clinical outcome suggesting promise for development of novel prognostic tests for cancer management and as aids in drug design.