Abstract
Abstract: :
Purpose: The purpose of this study is to investigate relative gene expression of angiogenic factors Cysteine rich protein (Cyr61), vascular endothelial growth factor (VEGF) and Tissue Factor (TF) in archival specimens of uveal melanoma utilizing real-time PCR. Methods: Reverse transcription of isolated RNA followed by kinetic quantitative polymerase chain reaction (real-time PCR) using gene specific primers to specific angiogenic factors was used to analyze mRNA expression in formalin-fixed paraffin-embedded (FFPE) samples. This comparatively new technique is highly sensitive and allows quantification of rare transcripts and small changes in gene expression. Tissue sections were de-paraffinized in xylene followed by re-hydration in 100-90-70% ethanol. The air dried tissue was then incubated at 60oC for 16 hours in 200µl of RNA lysis buffer containing 10mM Tris-Cl (pH 8.0), 0.1mM EDTA, 2% SDS (pH 7.3) and 500µg /ml of proteinase K. The clarified lysate was then extracted with water-saturated phenol-chloroform mix and the RNA was precipitated in the presence of 0.1volume of 3M NaOAc (pH 5.2), equal volume of iso-propanol and 20µg glycogen. After washing in 70% ethanol, the air dried pellet was dissolved in 10µl of water. cDNA was subjected to real-time PCR with gene specific primers for different angiogenic factors and normalized against the house keeping gene glucoronidase (GUS) using SYBR green incorporation for detection. Results: Different methods for isolation of RNA were tested and conditions optimized for FFPE specimens. Reproducibility of the method was tested using serial sections from the same specimen for RNA extraction followed by real-time PCR. Values for Cyr61expression, for example, varied between serial sections by less than 5%. Cloning of the products generated in real-time PCR reactions verified the fragments were GUS and Cyr61. Conclusions: It is now possible to use different FFPE specimens derived from patients with uveal melanoma to correlate different angiogenic gene expression with blood vessel density, other tumor parameters and patient outcome. Support NIH EY 12768 and EY 13705, Research to Prevent Blindness, Inc. ASP is a Jules &Doris Stein RPB Professor.
Keywords: melanoma • oncology • pathology techniques