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Updated: Apr 11, 2026
Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis
Published on: November 26, 2015
The PFA-AMeX method achieves a good balance between the morphology of tissues and the quality of RNA content in DNA
Takeshi Watanabe1, Atsuhiko Kato2, Hiromichi Terashima2
1Chugai Research Institute for Medical Science Inc., 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan.
Abstract:
Recently, large-scale gene expression profiling is often performed using RNA extracted from unfixed frozen or formalin-fixed paraffin embedded (FFPE) samples. However, both types of samples have drawbacks in terms of the morphological preservation and RNA quality. In the present study, we investigated 30 human prostate tissues using the PFA-AMeX method (fixation using paraformaldehyde (PFA) followed by embedding in paraffin by AMeX) with a DNA microarray combined with laser-capture microdissection. Morphologically, in contrast to the case of atypical adenomatous hyperplasia, loss of basal cells in prostate adenocarcinomas was as obvious in PFA-AMeX samples as in FFPE samples. As for quality, the loss of rRNA peaks 18S and 28S on the capillary electropherograms from both FFPE and PFA-AMeX samples showed that the RNA was degraded equally during processing. However, qRT-PCR with 3' and 5' primer sets designed against human beta-actin revealed that, although RNA degradation occurred in both methods, it occurred more mildly in the PFA-AMeX samples. In conclusion, the PFA-AMeX method is good with respect to morphology and RNA quality, which makes it a promising tool for DNA microarrays combined with laser-capture microdissection, and if the appropriate RNA quality criteria are used, the capture of credible GeneChip data is well over 80% efficient, at least in human prostate specimens.
Insights
The PFA-AMeX method offers superior morphological preservation and RNA quality for gene expression profiling compared to traditional FFPE methods. This technique is promising for DNA microarrays and laser-capture microdissection in prostate cancer research.
Area of Science:
- Molecular Biology
- Pathology
- Genomics
Background:
- Gene expression profiling commonly uses RNA from frozen or formalin-fixed paraffin-embedded (FFPE) samples.
- Both sample types present challenges in morphological preservation and RNA quality.
Purpose of the Study:
- To evaluate the PFA-AMeX method for its effectiveness in preserving morphology and RNA quality.
- To assess its suitability for DNA microarrays combined with laser-capture microdissection.
Main Methods:
- Investigated 30 human prostate tissues using the PFA-AMeX method.
- Utilized DNA microarray and laser-capture microdissection.
- Compared morphological preservation and RNA quality against FFPE samples.
Main Results:
- PFA-AMeX samples showed comparable morphological preservation to FFPE samples, clearly identifying basal cell loss in prostate adenocarcinomas.
- RNA degradation was observed in both PFA-AMeX and FFPE samples, but was less severe in PFA-AMeX.
- Quantitative RT-PCR indicated milder RNA degradation with PFA-AMeX, suggesting better RNA integrity.
Conclusions:
- The PFA-AMeX method provides excellent morphology and superior RNA quality for gene expression analysis.
- It is a promising technique for DNA microarrays coupled with laser-capture microdissection in prostate tissue studies.
- Credible GeneChip data capture exceeds 80% efficiency with appropriate RNA quality criteria.
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