Spot hybridization

Spotted arrays are routinely used to simultaneously analyze gene expression in two cell samples using one hybridization. In many cases, one of the two samples represents a control cell, providing an internally controlled reference for each spot hybridization. In con-... 

Fig. 3.46. " Sn image of Sn-labeled DNA hybridized to six oligonucleotide spots three complimentary and three non-comple-mentary.

Refers to the physical substrate to which biological samples are attached to create features (spots). In gene expression profiling arrays are hybridized with labeled sample and then scanned and analyzed to generate data. 

There are many protocols and different types of platforms available, e.g. GeneChips from Affymetrix, Illumina Bead Arrays, Arrays from Agilent, Applied Biosystems, GE Healthcare, customized spotted cDNA microarrays etc. the basic procedure for a large-scale measurement of gene expression involves the preparation of total or mRNA from the biological sample(s) under investigation (e.g. candidate tissue) and the hybridization of copied labelled RNA or cDNA to the DNA elements on the array surface (Fig. 1). 

A Petri dish containing bacterial colonies is blotted with nitrocellulose paper. This transfers a large portion of each colony to the paper, which is saturated with a solution that lyses (breaks open) the cells. The DNA of the lysed colonies is denatured with alkali. The nitrocellulose paper is neutralized, washed, and the paper either baked in an oven or treated with ultraviolet light to immobilize the denatured DNA. The DNA on the paper is hybridized with the labeled probe of interest, and the excess label is washed off. The dried paper is exposed to photographic film and the film developed. The exposed spots on the film can be matched with the colonies on the master plate and colonies picked off for further study. 

The data obtained has been derived from different experimental designs (namely, the number of collected fractions and the hybridization scheme), different microarray platforms (spotted versus Affymetrix arrays), and different data analysis schemes. In this chapter, we discuss various experimental designs for microarray analyses, provide detailed protocols for various experimental steps when using the spotted microarray platform, and discuss aspects of data analysis. 

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. 

Figure 3.3 Comparison of array CGH among DNA extracted from fresh tissue, FFPE tissue by heating protocol or nonheating protocol for two human tissue samples of metastatic carcinoma in lymph node (a-c), and undifferentiated non-small cell carcinoma (d-f). Array CGH hybridization genomic profiles show ratio values representing relative copy number of single BACs. A good result is scored as 1.0 that indicates a low standard deviation for gains (>0.2), normal (0.0), or losses (<-0.2). In these two cases, fresh samples show best score as 2, both FFPE tissue samples show identical score of 3. Each spot represents the average of three replicates. Clones are ordered by chromosomal position as numbers at the bottom (x axis) of each picture. The y axis is the log2 ratio of test reference intensity. Provided by Sandy DeVries from Dr. Frederic Waldman s Lab at UCSF.

The possibilities of measuring time-resolved hybridization in this parallel setup are shown in Figure 23, and it even allows to obtain kinetic information for all spots in parallel. Since reflectometric interference... 

Hybridized spots appear as grey spots in the scan. The darker the spot, the more target DNA is present. Kits based on this technology are currently under development by Eppendorf, Nanosphere and Clondiag. 

In a DNA array, gene-specific probes are created and immobilized on a chip (silicon wafer, nylon or glass array substrate). Biological samples are labeled with fluorescent dyes or radioactivity. These labeled samples are then incubated with the probes to allow hybridizations to take place in a high fidelity manner. After incubation, non-hybridized samples are washed away and spot fluorescent or radioactivity signals resulting from hybridization can be detected. 

Figure 11.15 (see Plate 5 for color version) shows ideal microarray spots. However, such ideal round spots are in fact impossible to achieve. As suggested by Yang et al. [28], the imperfect spot size and shape occurs during the hybridization and printing process and is also due to the conditions of the slide surface, making it more tedious to extract the raw data. Some of the imperfect microarray spots appear in the form of a comet tail , as shown in Fig. 11.16 (see Plate 6 for color version), which is due to... 

Figure 11 Chemiluminescent detection for membrane hybridization of unmodified DNA target by derivatization reaction with TMPG. Procedure [15] A portion of the DNA solution is spotted on a nylon membrane. The target DNA is hybridized to its cDNA probe having a -(G)15TT(G)15TT at its 3 terminus in a hybridization buffer (pH 7.0) at 42°C for 2 h. After washing, the membrane is moistened with sodium phosphate solution (pH 10) for a few seconds, and then immersed in 0.2 M TMPG dissolved with dimethyl sulfoxide for 0.5 min at ambient temperature. The moist membrane is then dipped in dimethyl-formamide for a few seconds, and the luminescence is detected for 0.5 min.

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