Microarray slide

Place a microarray slide in a hybridization chamber and pipette 25 Ail of the labeled probe mixture on the slide surface near one end of the microarray print area. 

Fig. 13.13 The number of probes on each tissue microarray slide can vary depending on the diameter of punched specimens...

Figure 2.2 Microarray slides (Photo courtesy of Thermo Fisher Scientific).

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. 

Novagen (ProteoPlex ), S S (FAST Quant), BioSource (Cartesian Array ), and BD Biosciences (BD Clontech Ab Microarrays) have introduced or will soon introduce protein microarray slide formatted products in which antibodies are directly immobilized. Beckman Coulter s protein array products for performing micro-ELlSAS in standard 96-well plate formats are based upon the self-assembly (by hybridization) of oligonucleotide-antibody conjugates to complementary oligonucleotides arrayed in individual wells. HTG s protein array technology was described previously. 

Cell microarrays have also been fabricated. Ziauddin and Sabatini (2001) demonstrated the ability to transfect cells cultured onto plasmid DNA arrayed in gelatin on a standard DNA microarray slide. Xu (2002) printed down cells in the form of high density microarrays on permeable membranes and demonstrated phenotypic assay performance with the immobilized cells. The commercialization of viable cell arrays will permit an even closer look at cell-mediated events during the drug discovery process. 

The primary substrate for spotted arrays is the glass slide. The salient physical and chemical features of a microarray slide are optical clarity (including... 

Others such as Macas et al. (1998) successfully adapted the Biomek 2000 (Beckman Coulter), a commonly used liquid handling robot, to prepare microarray slides using a specially constructed print head and quill pins. Up to 28 microscope slides could be placed on a work surface for printing. Biomek s HDRT head was adapted to accept microarray quill pins held between two parallel plates with holes drilled on 9-mm centers to dip into 96-well source plates. The quill pins were spring-loaded similar to the design... 

Slide-based microarray technology was first introduced by Schena etal. (1995). The processes and equipment for preparing (arrayer) and analyzing (laser scanner) microarray slides comprised a portion of Dari Shalon s thesis work at Stanford University. Polymerase chain reaction (PCR) products (cDNA probes) were attached to PLL-coated glass microscope slides. The... 

In terms of sensitivity, the detection limits on the microarray slide using the tw o-color system approached 0.1 ng/mL for antigen arrays and 1 ng/mL for antibody arrays. Both were able to measure specific proteins in mixtures at 1 part per million in total protein (partial concentration). 

Figure 10.2 This scientist is examining a microarray slide. The pattern of binding of the test samples, to the array of DNA on the slide, is displayed on the computer screen. Libraries of small chemicals can also be analyzed for their ability to bind to DNA or protein microarrays in a search for potential new drugs.

Preblock the microarray slide with kinase buffer and 0.1% BSA, 30 min. 

Rinse printed microarray slides with IX PBS (pH 7.4) with 0.05% Tween-20 for 5 min. 

One Microarray Slide - - 40 MB One full length music album... 

The vast majority of array assays have used fluorescence as the detection method due to the high sensitivity and the availability of detectors such as fluorescent plate readers for microtiter plates and high resolution DNA microarray scaimers for glass microarray slides. Therefore, the following discussion will focus on assay development coupled with fluorescent detection. 

Figure 2.2 Microarray slides Photo courtesy of Affymetrix. Inc., US.

After the microarray slides were hybridized with labeled cDNAs derived from these 5 populations of RNAs, the data were analyzed by several different comparisons. 

Selection of a number of cases from patient subsets in a given microarray slide is amenable to statistical modeling to enhance analysis of results. Different microarrays can be constructed to answer different scientific questions. The microarrays can also be produced from archival material, using paraffin blocks of already characterized tumors with clinical follow-up. This would provide a rapid evaluation of clinically well-characterized tumors. The viability of this approach has been tested with prostate carcinomas, renal cell carcinomas, and other tumors (5,6). 

The limited volume of the total cellular material from biopsy samples makes it necessary to use a slight modification of the lysis buffer recipe as compared with the cell culture lysis buffer. The following protocol describes a method for preparing whole cell lysates from LCM cells. The limited biopsy sample generally precludes the use of total protein assays before microarray printing. To compensate for this, one microarray slide is stained for total protein using a Sypro Ruby Protein blot stain (see Subheading 3.3.3.). 

Each microarray is probed with a single primary antibody directed against the protein of interest. Microarray slides used for immunostaining should be blocked before staining. Microarray slides used for Sypro Ruby protein staining do not require blocking. 

Remove microarray slide(s) from freezer and leave at room temperature for approximately 5-10 min. 

Prepare a lx solution of Reblot mild solution in deionized water. Wash microarray slides with gentle rocking in lx Reblof mild solution for 15 min see Note 10). 

Immunostaining requires the use of control slides for determining background staining due to secondary antibody alone. Each species-specific secondary antibody should be used to stain an individual microarray. Therefore, for any immunostaining run, one slide must be used as a negahve (secondary antibody alone) control slide. The total number of slides to be stained is determined by the number and species of primary antibodies. For example, if three anti-rabbit primary antibodies and one mouse primary antibody are selected, a total of 6 microarray slides will be needed (4 antibodies + 2 controls). 

Load reagents and microarray slides on the Autostainer (see Note 13). 

The limited sample material from microdissected biopsy lysates precludes the use of spectrophotometeric analysis of total protein prior to microarray printing. A microarray slide stained for total protein serves as a tool for normalizing spot intensity between samples (see Note 15). 

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