title: Gene transcript quantitation by real-time RT-PCR in cells selected by immunohistochemistry-laser capture microdissection.
abstract: Studying tissue-based gene expression in different cell populations often requires immunohistochemistry-guided microdissection. However, mRNA degradation occurs during long staining procedures. We combined a novel rapid immunoperoxidase technique with laser capture microdissection (LCM) and real-time quantitative RT-PCR to compare p27 mRNA expression in prostatic basal/secretory cells. Eight frozen prostate sections were immunostained with antibody 34betaE12 (high-molecular-weight keratin). Secretory and basal cells were separately collected by LCM. p27 transcripts from each cell group were quantitated by real-time RT-PCR, with GAPDH as standard. Immunostaining took 22 minutes, with RNA extraction from approximately 40 dissected cells from each compartment initiated within 40 minutes. Qualitative RT-PCR gave a product of the expected size from each sample. Quantitative RT-PCR gave basal/secretory p27/GAPDH ratios of 0.99-16.24 (mean 5.53 +/- 0.643). Immunostaining for keratin 34betaE12 can be done on frozen sections in approximately 20 minutes, and mRNA from pure cell populations can be quantitated by RT-PCR. We used this technique to show that p27 transcript levels are greater in basal than in secretory prostate cells, suggesting, when combined with prior studies, that regulation of p27 occurs at the protein level in normal cells. This technique may have wide applicability to studies of gene expression in distinct cell populations in heterogeneous tissues.
authors.affiliation: Department of Pathology, Brigham & Women's Hospital, Boston, MA 02115, USA.
authors.forename: Neal
authors.initials: N
authors.lastname: Lindeman
authors.forename: David
authors.initials: D
authors.lastname: Waltregny
authors.forename: Sabina
authors.initials: S
authors.lastname: Signoretti
authors.forename: Massimo
authors.initials: M
authors.lastname: Loda
mesh_terms: D004210:Dissection / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D007834:Lasers; D008297:Male; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis; D011467:Prostate / Q000166:cytology* / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; C413374:p27 antigen