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.
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 stromal 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.
Pinzani
Pamela
P
Department of Clinical Physiopathology, Clinical Biochemistry Unit, University of Florence, Italy.
Lind
Kristina
K
Malentacchi
Francesca
F
Nesi
Gabriella
G
Salvianti
Francesca
F
Villari
Donata
D
Kubista
Mikael
M
Pazzagli
Mario
M
Orlando
Claudio
C
D000230:Adenocarcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000911:Antibodies, Monoclonal / Q000276:immunology; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007834:Lasers; D008297:Male; D042282:Microdissection; D016133:Polymerase Chain Reaction / Q000379:methods; D011467:Prostate / Q000378:metabolism / Q000473:pathology; D017430:Prostate-Specific Antigen / Q000235:genetics* / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D017430:Prostate-Specific Antigen