Microarrays sectioning

The cDNA technology is essentially an array-based, high-throughput protocol that determines gene expression and copy number survey of very large numbers of tumors. As many as 1,000 cylindrical tissue biopsies from individual tumors can be distributed in a single tissue microarray. Sections of the microarray also provide targets for parallel in situ... 

Figure 10.1 Experimental schemes for microarray analysis. All experimental schemes start with a separation step of the cell lysate by velocity sedimentation in a sucrose gradient (top scheme). Collection of the desired fractions is assisted by a continuous ultraviolet (UV) reading of the gradient (an example of such UV reading is shown in each section). This allows determination of the sedimentation position of the 40S, 60S, 80S, and polyribosomal complexes (2,3, and more).Three general ways for fraction collection and analysis are presented (sections A, B, and C) (A) Collection of two fractions (free and polysomes) and direct comparison between them, with the free mRNA fraction labeled with green dye and the polysome fraction labeled with red dye. (B) Collection of two fractions and indirect comparison between them by utilizing an unfractionated reference RNA. (C) Collection of multiple fractions (four in this case), where each fraction is compared to an unfractionated reference sample. The blue arrows indicate the addition of spike-in RNA to each fraction and to the reference RNA.

The DNA microarrays are spotted on glass slides coated with amino-silane (Coming GAPS II). They should be ready for hybridization immediately when the labeled cDNA is ready. Thus, while the dyes are couplingto the cDNA (step 3 in the previous section), it is recommended to start the following process. 

These tissue cores are then inserted in a recipient paraffin block (Fig. 13.12) in a precisely spaced array pattern. Each microarray block can be cut into from 100 to 1000 sections (Fig. 13.13), which can be subjected to independent tests with immunolabeling (Moch et al. 2001). 

This section provides only a basic outline of concepts surrounding cDNA microarrays. Eor further reading a number of excellent detailed descriptions of this technology are recommended (Geschwind, 2000 Luo and Geschwind, 2001 Marcotte et al., 2001 Li et al., 2002). 

The typical cDNA microarray study can be described in nine steps (1) establishing an appropriate experimental design (2) isolation and conversion of mRNA to labeled cDNA (3) hybridization of labeled cDNA to the microarray slide (4) image acquisition, (5) data storage, (6) normalization (7) statistical analysis (8) data mining and (9) validation of the results. Each of these steps is multifaceted and the introduction of error at any point in the process can lead to costly loss of data. The following section describes the steps followed in experimental design. 

Sandwich assay — This format works well for ELISA. The success and potential shortcomings for microarrays are discussed in the next section. 

In the following sections, the major types of substrates currently used for DNA and protein microarrays will be discussed. Much of what is known regarding microarray surface chemistry and the immobilization of biomolecules comes from work with DNA microarrays. Therefore, many of the examples cited here will be from these studies. Zhu and Snyder (2003) in their review provide good insight into the manufacture and utility of protein microarrays. Here are some points to consider when choosing a substrate for protein microarrays ... 

In the following sections, we will look at representative applications of DNA microarrays in the biomedical research field. 

Based on transcriptional profiling, Oncotype DX and MammaPrint, which will be described in details later in this section, have been developed and used in clinics. It has been shown that the gene expression-based microarray profiling offers tremendous potential to define subcategories of breast cancer, to predict disease relapse, to predict chemotherapy response, and to predict progression of ductal carcinoma in situ (7). 

The chapter is divided into four complementary sections. Sections 2 to 4 describe the process of sequence analysis, data management and reporting standards, and the design and analysis of microarray experiments. To demonstrate the described computational tools and concepts covered in these sections, a practical example is illustrated in Section 5. 

This section will discuss the standards for sharing and discussing microarray experiments, including the ontologies and controlled vocabularies. A non-technical description of the sharing process will be provided, as well as an introduction to the pharmacology/toxicology focused version of the MIAME standard, MIAME/Tox, which is currently under development. 

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