title: The PFA-AMeX method achieves a good balance between the morphology of tissues and the quality of RNA content in DNA microarray analysis with laser-capture microdissection samples.
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.
authors.affiliation: Chugai Research Institute for Medical Science Inc., 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan.
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Kato
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Terashima
authors.affiliation: PharmaLogicals Research, Pte. Ltd., 11 Biopolis Way, #05-08/09 Helios, 138667, Singapore ; Chugai Pharmabody Research Pte. Ltd., 3 Biopolis Drive, #04-11 to 17 Synapse, 138623, Singapore.
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsubara
authors.affiliation: PharmaLogicals Research, Pte. Ltd., 11 Biopolis Way, #05-08/09 Helios, 138667, Singapore ; Chugai Pharmabody Research Pte. Ltd., 3 Biopolis Drive, #04-11 to 17 Synapse, 138623, Singapore.
authors.forename: Yu Jau
authors.initials: YJ
authors.lastname: Chen
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Adachi
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mizuno
authors.affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Suzuki
keywords: AMeX method; DNA microarray; PFA; RNA quality; laser-capture microdissection; prostate