DNA Microarrays and Gene Expression

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Hybridization between the cDNA reverse transcribed from a biological sample to a pre-designed complementary DNA probe arranged on a slide, or array, is the basis of DNA microarrays. A microarray therefore consists of a pre-designed library of synthetic nucleic acid probes that are immobilized and spatially arrayed on a solid matrix. Microarrays evolved from a technique known as Southern blotting , where DNA fragments are attached to a substrate and then probed with a known gene sequence.

There are several ways that such spotted arrays can be produced. Other methods employ photo-activated chemistry and masking to synthesize probes one nucleotide at a time on a solid surface in repeated steps to build up probes of specific sequence in designated locations. The resulting "grid" of probes can hybridize to complementary "target" sequences derived from experimental samples to determine the expression level of specific mRNAs in a sample. If we are trying to calculate relative expression between two samples, each labeled with a different dye See figure 2, red for experiment, green for control , the resulting image is analyzed by calculating the ratio of the two dyes.

If a gene is over-expressed in the experimental sample, then more of that sample cDNA than control cDNA will hybridize to the spot representing that expressed gene. In turn, the spot will fluoresce red with greater intensity than it will fluoresce green. The red-to-green fluorescence ratio thus indicates which gene is up or downregulated in the appropriate sample.

Figure 2: A typical DNA microarray co-hybridization 2 dye experiment. Microarray technology propelled functional genomics, a discipline that strives to identify the role of genes in cellular processes, into the spotlight because it allowed functional analysis of genome-wide differential RNA expression between different samples, states and cell types to gain insights into molecular mechanisms that regulate cell fate, development, and disease progression.

Microarray data is used to generate a profile of gene expression, which serves as a determinant of protein levels and therefore cellular function between biological samples.

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A technician applies the label and serial number to a GeneChip cassette. The GeneChip is now ready to be used in a laboratory to perform large-scale genetic analysis. Doctor Josh Dubnau explains that some genes are preferentially active in one part of the brain or body, while other genes are particular active in another location. Professor David Lewis outlines how microarrays have transformed the search for schizophrenia genes and led to his group's discovery of the candidate gene, RGS4.

Download MP4. Related Content. DNA microarray output DNA microarrays provide the means to analyze patterns of gene expression at different timepoints in a living cell.

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Animation The RNA message is sometimes edited. Rich Roberts and Phil Sharp explain restriction enzymes, electrophoresis, and split genes. The DNA microarray technique constitutes the most promising and revolutionary ever-developed technique to study differential gene expression.

In this work, the DNA microarrays technique was applied to X. Genomic DNA extraction was carried out with X. The genomic DNA was extracted according to the methodology described by Ausubel et al. The RNAs samples were obtained from X. The DNA integrity was analyzed by electrophoresis in 1. RNA integrity was verified in a 1.

Introduction

The CyDye incorporation efficiency was monitored by absorbance measurements at different wavelengths: nm for DNA concentration , nm for Cy3 and nm for Cy5. Microarray construction. Specific primer pairs were designed for the whole X. These primer pairs were used to amplify the 2, ORFs of X. All products were analyzed by electrophoresis in 1. Genetically distant negative controls were also included in this array with human pHUM1 and pHUM7 and plant genes B11 -Rubisco , as well as synthetic controls from various species, such as: human, mouse, leaven, Arabidopsis spp.

Hybridization and Washing. This solution was injected in the hybridization chamber to cover the arrays.

A DNA microarray survey of gene expression in normal human tissues

All the washing steps consisted of 10 cycles of solution flow 10 s and incubation 20 s. The slides were dried for 15 min and submitted to fluorescence detection. Image acquisition and data analyses. The signal of each spot was quantified with the ImaGene software v. Using DNA microarrays for genomic analysis of Xylella fastidiosa.

Construction of DNA microarrays. Therefore each of the X. The amplifications were considered successful when only one product was visualized, within a size range of 0. The minimum required DNA concentration depends on some factors, such as probe length, base composition and binding capacity of the arrays substrate Deyholos and Galbraith, The effect of probe length 0. Human pHUM1 and pHUM7 and plant genes BRubisco , as well as synthetic genes from several species human, mouse, rat, yeast, plant and bacteria were also included in this array as negative controls.

These controls play an important role in microarray data analysis because they allow signal levels evaluation from nonspecific hybridization.

DNA Microarray synthesis

Any spot on the array, which presents a signal not significantly stronger than the one from the negative control, should be scored as absent from the fluorescently labeled target Holloway et al. The X. Studies on microarray gene expression analysis, using non purified amplified products, have shown no significant differences between the purified and non purified PCR products. The amplicons were printed on silane-coated glass microscope slides by an Affymetrix Arrayer.

A fraction of the ring fluid is transferred onto the microscope slide when the pins pass through the sample and push a tiny drop to the surface. This robot is designed to collect samples from 96 or well microtitre plates, with four pins and rings simultaneously. Each ring collects 0. However, the size of the resulting spot is a function of the pin diameter, the pin material, the fluid viscosity, and the dynamics of interaction between the fluid and the surface. For this reason, aqueous solutions deposited on hydrophobic or hydrophilic surfaces spread differently before evaporation Mace et al.

The main advance of DNA microarray technology arises from the array small size, which allows higher sensitivity, enables the screening of a larger number of genes and provides the opportunity to use smaller amounts of starting material, compared to conventional techniques van Hal et al. Southern et al. However, some of the common problems found in glass slides are due to the spot morphology and the high background, which may be related to the batch variability Ye et al.

Other problems associated to the irregular spot morphology were pin defects, low humidity throughout the printing and low DNA concentration. As quoted elsewhere, the problem of DNA concentration was corrected by gel analysis in order to quantify the amplified DNA, immediately prior to the printing.

Profiling C. elegans gene expression with DNA microarrays

Finally, the change of the pins for new ones had to be solved. The pins were always cleaned at the end of each cycle, allowing the same set of pins to be used in different array without cross-contamination. The hypothesis of pins damage in contact with the glass slide is strongly supported by variations in height between the slides. For this reason, both the pin strength and speed must be corrected in order to avoid its impact on deformation and the spot morphology modification.


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  • Before robotic printing, some geometric tests were performed to determine the best spot distribution on the slides data not shown. The linear geometry is an excellent standard for the printing of many samples plates and their replicas. However, the array geometry is used to control the localization of these replicates and the spaces in the slides, so that a small volume is necessary to the hybridization. In this experiment, the spots were printed in arrays of 4 x 2. After spotting, the arrays were submitted to a heat treatment for both DNA sequences attaching to the glass surface and denaturation.

    The spotted amplicons were re-hydrated, dried and fixed in a cross-linked UV camera. The used slides were coated with gamma-amino propyl silane because it limited the spotted DNA droplet dispersion and enhanced the slides hydrophobicity, also increasing the adherence of the deposited DNA Duggan et al. DNA is bound to the substrate through the electrostatic interaction between the silane amine groups.

    The positively charged primary amines attract the DNA negatively charged phosphodiester backbone Stillman and Tonkinson, UV-irradiation enhances and stabilizes such interaction by generating free-radical-mediated coupling of thymidine residues and carbon atoms of the alkyl amine Holloway et al. Synthesis of fluorescent labeled cDNA. Preparation of fluorescent labeled cDNAs was carried out by total RNA extraction, and its concentration was determined by absorbance measurement at nm A The quality of RNA is critical factor for microarray analysis. The extraction and purification steps are particularly critical since they can cause RNA contamination, which can mediate significant non-specific binding of fluorescently labeled cDNAs to the slide surface Duggan et al.

    The RNA integrity was checked by formaldehyde agarose gel electrophoresis, where the occurrence of the two ribosomal subunit bands 23S and 16S containing of 2. The fluorescent labeled cDNAs were prepared from total X. Lucchini et al. However, no satisfactory results was obtained with the synthesized primers for X.

    A limitation of this technology is the large amount of RNA required for hybridization. According to Ye et al. Labeling efficiency by reverse transcription depends on the incorporation efficiency and on the amount of specific nucleotides present in a particular mRNA species. The used labeling kit was developed as a two-step procedure.



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