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.

Keywords: AMeX method; DNA microarray; PFA; RNA quality; laser-capture microdissection; prostate.

Fig. 1.

Schematic drawings of the assay concept for evaluating RNA quality by qRT-PCR using 3’and 5’ primer sets for beta-actin. Based on the assay concept, the amounts of 3’ and 5’ amplicons will be close to equal in high-quality RNA samples, but the amount of 5’ amplicon will be decreased in low-quality RNA samples. Black bar: mRNA. Red bar: primer sets.

Fig. 2.

Histological features of prostatic adenocarcinoma Gleason patterns 3 (a, b, c), and 4 (d, e, f). In adenocarcinomas with Gleason pattern 3, the basal cells were indistinct in F-F specimens (a) and were clearly identified in glands adjacent to adenocarcinoma in FFPE (b) and PFA-AMeX specimens (c). In adenocarcinomas with Gleason pattern 4, cancer cells were clearly distinguishable from the noncancerous and surrounding connective tissues of F-F (d), FFPE (e) and PFA-AMeX specimens (f). AC, adenocarcinoma. Arrows, distinguishable basal cells. H&E staining: bar = 50 μm.

Fig. 3.

RNA quality of xenograft tissues evaluated with a bioanalyzer. Typical capillary electropherograms of total RNA from F-F (a), FFPE (b) and PFA-AMeX (c) samples of LNCaP xenograft tissue.

Fig. 4.

RNA quality of xenograft tissues evaluated by measuring beta-actin mRNA. Log scale scatter plot for 5’ amplicon amounts and 3’/5’ ratios of beta-actin in F-F, FFPE and PFA-AMeX samples of LNCaP xenograft tissue.

Fig. 5.

RNA quality of human prostate samples evaluated by measuring beta-actin mRNA. Log scale scatter plot for 5’ amplicon amounts and 3’/5’ ratios of beta-actin from FFPE and PFA-AMeX samples of human prostate tissue. The Shaded area shows the cutoff line for RNA quality (5’ amplicon ≥0.5, 3’/5’ ratio ≤20).

Fig. 6.

Evaluating the quality of GeneChip human prostate data. Scatter plot of %Pcall and SF of GeneChip data for human prostate tissue from FFPE and PFA-AMeX samples. The shaded area shows the sector that matched the GeneChip QC criteria (%Pcall ≥30, SF ≤1.5).