An easy setup for double slide microarray hybridization
| Autor: | Shern-Fwu Lee, Kai Wang, Abigail C. Ting |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Microscope
Oligonucleotide Microscope slide Reproducibility of Results Equipment Design Biology Molecular biology Sensitivity and Specificity General Biochemistry Genetics and Molecular Biology law.invention Equipment Failure Analysis Fluorescent labelling law Gene chip analysis Microarray hybridization Hybridization Genetic Single probe DNA microarray Biological system DNA Probes Biotechnology Oligonucleotide Array Sequence Analysis |
| Zdroj: | BioTechniques. 35(4) |
| ISSN: | 0736-6205 |
| Popis: | Since the inception of microarray technology, the amount of available gene expression information has sky-rocketed. As a massive parallel plat-form for data gathering, microarrays make it possible to obtain data about global gene expression changes under varying physiological or pathological conditions (1−3). Gene probes labeled with fluorescent dye, along with mi-crospotting devices, make microarray technology a sensitive and high-throughput tool for measuring expres-sion level changes of tens of thousands of transcripts simultaneously (4). A microarray experiment gener-ally begins with gene targets (cDNA or oligonucleotides) spotted onto a pro-cessed microscope slide. A coverslip is then used to distribute the hybridization solution across the spotted area (array). Finally, the setup is placed in a humid hybridization chamber to maintain a stable microenvironment during hybridization. While microarray tech-nology offers enormous increases in throughput, data quality still depends on various experimental conditions. These include the quality of printed DNA and processed microscope slides, environmental controls during printing, composition and temperature of hybrid-ization solution, and the quality of the probes. All of these factors influence reproducibility, and often an experi-ment must be repeated numerous times to increase the data reliability (5−8).Two-color fluorescent labeling has made microarray technology more powerful than the traditional single-labeling hybridization procedure. However, a drawback of this fluores-cence-based technology is that fluo-rescent probes are less sensitive than radioactively labeled ones (9). For this reason, microarray experiments gener-ally require large amounts of RNA as the initial labeling material (sometimes up to 100 µg; see protocol in http://cmgm.stanford.edu/pbrown/protocols/4_human_RNA.html). This can lead to difficulties when dealing with clinical samples, where the samples are often small and hard to obtain. It limits the potential for repeating experiments to increase the reliability of the data or probing different gene arrays to expand the gene coverage. One way to alleviate these problems is to hybridize multiple slides using a single probe preparation. We are aware of one commercially developed double slide hybridization setup. In this ar-rangement, two arrayed slides are set face-to-face inside a chamber and se-cured by an attached comb-like clamp along each long edge of the unit. The teeth of the comb maintain space be-tween the two slides. The hybridization solution can then be pipetted between the teeth and is drawn inside by capil-lary action. This commercially avail-able product, while rather expensive, addresses the possibility of increasing gene coverage within a single hybrid-ization procedure. As a cost-saving measure, we have worked to develop our own double slide hybridization approach. We have experimented with sandwiching the hybridization solution directly be-tween two slides. A scan of an on-line microarray discussion group (http://groups.yahoo.com/group/microarray) turns up discussion from others on their efforts with this method. However, there are several disadvantages to the direct sandwich system. First of all, it is difficult to move or handle the setup once the slide pair is assembled with the hybridization solution. Second, contour variations of a slide surface affect the distribution of solution and the amount needed, making it difficult to estimate the required volume of hybridization solution. Third, probes may be diluted, since the volume of the hybridization buffer has to increase to cover the entire slide rather than only the arrayed area, compared to using a coverslip. Finally, the weight of the top slide may impede the flow of hybrid-ization buffer and cause uneven and irreproducible results.In this paper, we report on a reliable, easy, and inexpensive double slide hybridization setup that eliminates |
| Databáze: | OpenAIRE |
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