Gene transcript quantitation by real-time RT-PCR in cells selected by immunohistochemistry-laser capture...

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Studying gene expression in specific cell types within tissues is challenging due to mRNA degradation during lengthy staining processes.
• Immunohistochemistry-guided microdissection is often required but can compromise RNA integrity.

Purpose of the Study:

• To develop and validate a rapid technique combining immunoperoxidase staining, laser capture microdissection (LCM), and quantitative RT-PCR for analyzing gene expression in distinct cell populations.
• To compare p27 mRNA expression levels between basal and secretory cells in the prostate.

Main Methods:

• A novel, rapid immunoperoxidase staining technique was employed on frozen prostate sections using an antibody for high-molecular-weight keratin (34betaE12).
• Specific cell populations (basal and secretory) were isolated using laser capture microdissection (LCM).
• RNA was extracted, and p27 mRNA expression was quantified using real-time quantitative RT-PCR, with GAPDH as an internal standard.

Main Results:

• The rapid immunostaining procedure took approximately 20 minutes, allowing RNA extraction within 40 minutes.
• Quantitative RT-PCR confirmed the presence of p27 transcripts in both cell types.
• p27 mRNA levels, normalized to GAPDH, were significantly higher in basal cells compared to secretory cells (mean ratio 5.53).

Conclusions:

• This rapid technique enables the reliable quantitation of mRNA from pure cell populations isolated from heterogeneous tissues.
• The findings demonstrate higher p27 transcript levels in prostatic basal cells versus secretory cells.
• This suggests that p27 regulation may occur at the protein level in normal prostate cells and highlights the technique's broad applicability.