A general framework for designing and validating oligomer-based DNA microarrays and its application to Clostridium acetobutylicum.

While DNA microarray analysis is widely accepted as an essential tool for modern biology, its use still eludes many researchers for several reasons, especially when microarrays are not commercially available. In that case, the design, construction, and use of microarrays for a sequenced organism constitute substantial, time-consuming, and expensive tasks. Recently, it has become possible to construct custom microarrays using industrial manufacturing processes, which offer several advantages, including speed of manufacturing, quality control, no up-front setup costs, and need-based microarray ordering. Here, we describe a strategy for designing and validating DNA microarrays manufactured using a commercial process. The 22K microarrays for the solvent producer Clostridium acetobutylicum ATCC 824 are based on in situ-synthesized 60-mers employing the Agilent technology. The strategy involves designing a large library of possible oligomer probes for each target (i.e., gene or DNA sequence) and experimentally testing and selecting the best probes for each target. The degenerate C. acetobutylicum strain M5 lacking the pSOL1 megaplasmid (with 178 annotated open reading frames [genes]) was used to estimate the level of probe cross-hybridization in the new microarrays and to establish the minimum intensity for a gene to be considered expressed. Results obtained using this microarray design were consistent with previously reported results from spotted cDNA-based microarrays. The proposed strategy is applicable to any sequenced organism.

Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts.

The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) technique has been extensively used for the detection and quantification of apoptosis in histological tissue sections. However, the interpretation and specificity of this assay have been controversial. With accumulating knowledge of the molecular mechanisms of cell death and the discovery of the caspases as key mediators of apoptosis, more direct and earlier measurements of apoptosis in tissue sections have emerged. This study, using antibodies that specifically recognize activated caspase-3 and caspase-cleaved cytokeratin (CK) 18, evaluated whether immunohistochemical stains would improve the detection and quantification of apoptosis in tissue sections, compared with the TUNEL assay. Tumour xenografts of the prostate cancer cell line PC-3 were used as an example, since these tissues contain large numbers of cells undergoing apoptosis. Apoptotic cells were quantified and apoptotic indices were calculated by computer-assisted image analysis following identification of apoptotic cells by morphological analysis, the TUNEL assay, activated caspase-3 and cleaved CK18 immunohistochemistry. The results indicated that activated caspase-3 immunohistochemistry was an easy, sensitive, and reliable method for detecting and quantifying apoptosis in this model. An excellent correlation (R = 0.89) between the apoptotic indices obtained using activated caspase-3 and cleaved CK18 immunostaining was observed. A good correlation (R = 0.75) between the apoptotic indices obtained using activated caspase-3 immunostaining and the TUNEL assay was also found. Activated caspase-3 immunohistochemistry is therefore recommended for the detection and quantification of apoptosis in tissue sections.