Optimized RNA Extraction from Non-deparaffinized, Laser-Microdissected Material
mRNA extraction and subsequent RT-polymerase chain reaction (PCR)-based expression analysis from laser-microdissected material is by now a well-established and reproducible method. Most routinely stored tissue samples are preserved as formalin-fixed, paraffin-embedded materials. While this allows for a convenient storage and stable preservation of nucleic acids, deparaffinization before staining for laser microdissection may result in a significant loss of mRNA quality and consequently of PCR sensitivity. We describe a method of isolating anatomic compartments from non-deparaffinized, formalin-fixed, and paraffin-embedded tissues by laser-assisted microdissection which allows for a highly efficient mRNA retrieval.
Available from: Takeshi Watanabe
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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.
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ABSTRACT: Chronic lung allograft dysfunction (CLAD) is the main reason for poor long-term outcome of lung transplantation, with bronchiolitis obliterans (BO) representing the predominant pathological feature. BO is defined as a progressive fibrous obliteration of the small airways, thought to be triggered by a combination of nonimmune bronchial injury and alloimmune and autoimmune mechanisms. Because biopsy samples are too insensitive to reliably detect BO and a decline in lung function test results, which is clinically used to define CLAD, does not detect early stages, there is need for alternative biomarkers for early diagnosis. Herein, we analyzed the cellular composition and differential expression of 45 tissue remodeling-associated genes in transbronchial lung biopsy specimens from two cohorts with 18 patients each: patients who did not develop CLAD within 3 years after transplantation (48 biopsy specimens) and patients rapidly developing CLAD within the first 3 postoperative years (57 biopsy specimens). Integrating the mRNA expression levels of the five most significantly dysregulated genes from the transforming growth factor-β axis (BMP4, IL6, MMP1, SMAD1, and THBS1) into a score, patient groups could be confidently separated and the outcome predicted (P < 0.001). We conclude that overexpression of fibrosis-associated genes may be valuable as a tissue-based molecular biomarker to more accurately diagnose or predict the development of CLAD.
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