A ribonuclease protection assay-based approach for analysis of angiogenic gene expression in archival tissues.
Archival, formalin-fixed and paraffin-embedded tissues routinely stored in pathology departments represent an invaluable resource for retrospective molecular biology studies for diagnostic and prognostic purposes. In such specimens extraction of transcriptionally competent RNA to be analyzed by conventional techniques, such as reverse transcription-polymerase chain reaction, is a challenging task. Therefore, we developed a novel methodological approach that allows successful detection and semiquantitative analysis of specific mRNAs obtained from archival formalin-fixed, paraffin-embedded specimens by ribonuclease protection assay. Specifically, we measured a panel of 7 angiogenic markers in selected archival tissues stored at room temperature and retrieved over a wide time span (10 y). The study series consisted in samples of benign and malignant melanocytic lesions. In our model, expression of FLT-1, the vascular-endothelial growth factor receptor-1, correlated with the expression of mRNAs encoding other tyrosine kinase receptors, such as TIE-1 and TIE-2, as well as with angiopoietin and with the protease-activated receptor-1 and vascular-endothelial growth factor itself. Relative to control (normal skin), in melanoma the expression of the selected angiogenic markers was significantly higher. In conclusion, our study provides evidence that ribonuclease protection assay on archival specimens would be highly valuable for retrospective studies, for diagnosis or prognosis.
Naldini
Antonella
A
Department of Physiology, University of Siena, Italy. Naldini@unisi.it
Ardinghi
Camilla
C
Massi
Daniela
D
Santucci
Marco
M
Franchi
Alessandro
A
Tarantini
Francesca
F
Geppetti
Pierangelo
P
Cirino
Giuseppe
G
Carraro
Fabio
F
D018070:Biological Specimen Banks; D005557:Formaldehyde; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D008545:Melanoma / Q000098:blood supply / Q000235:genetics; D009389:Neovascularization, Pathologic / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000302:isolation & purification*; D012260:Ribonucleases; D012871:Skin Diseases / Q000235:genetics; D012878:Skin Neoplasms / Q000098:blood supply / Q000235:genetics; D016707:Tissue Fixation
D012333:RNA, Messenger; D005557:Formaldehyde; D012260:Ribonucleases