Prostate-specific antigen mRNA and protein levels in laser microdissected cells of human prostate measured by real-time reverse transcriptase-quantitative polymerase chain reaction and immuno-quantitative polymerase chain reaction
Artikel i vetenskaplig tidskrift, 2008

Laser-assisted microdissection has mainly been used in cancer studies to excise pure cell populations from heterogeneous tissues. Cancer and normal cells selected by laser-assisted microdissection have frequently been used for mRNA expression studies usually by reverse transcriptase-quantitative polymerase chain reaction (qPCR). Recently, real time immuno-qPCR was developed as a new tool for highly sensitive measurements of proteins. Using reverse transcriptase-qPCR and immuno-qPCR, we measured the amounts of prostate-specific antigen mRNA and its corresponding protein in homogeneous and comparable cell populations, collected from normal and cancer prostates by laser-assisted microdissection. With these techniques, prostate-specific antigen mRNA and protein were quantified over a wide range of concentrations with a sensitivity sufficient to analyze single prostate cells (LNCaP). We did not find significant differences in prostate-specific antigen protein and mRNA between normal and cancer cells. The expression of prostate-specific antigen protein and mRNA was highly correlated in both normal and pathological cells. In microdissected peritubular stromas areas of prostate cancers, the concentration of prostate-specific antigen protein was about 100 times higher than in normal prostate, indicating an increased transit of secreted prostate-specific antigen. In the same samples, prostate-specific antigen mRNA was not detectable. Our data demonstrate, for the first time, the feasibility of simultaneous application of reverse transcriptase-qPCR and immuno-qPCR in studies of homogeneous cell populations, collected by laser-assisted microdissection. The approach is expected to become a very powerful tool for expression studies in human cancers at both mRNA and protein levels.

Författare

P. Pinzani

Universita degli Studi di Firenze

Kristina Lind

Chalmers, Kemi- och bioteknik, Molekylär mikroskopi

F. Malentacchi

Universita degli Studi di Firenze

G. Nesi

Universita degli Studi di Firenze

F. Salvianti

Universita degli Studi di Firenze

D. Villari

Universita degli Studi di Firenze

Mikael Kubista

Chalmers, Kemi- och bioteknik, Molekylär mikroskopi

M. Pazzagli

Universita degli Studi di Firenze

C. Orlando

Universita degli Studi di Firenze

Human Pathology

0046-8177 (ISSN)

Vol. 39 10 1474-1482

Ämneskategorier

Industriell bioteknik

DOI

10.1016/j.humpath.2008.02.012