Analysis mRNA microarray

Abbreviation Bp, nucleotide base pairs cDNA, complementary DNA ChIP, chromatin Immunoprecipi-tation Cy5, cyanine 5-dCTP Cy3, cyanine 3-dCTP ESTs, expressed sequence tags FDR, false discovery rate MIAME, minimum information about a microarray experiment mRNA, RNA, messenger NIA, National Institutes of Aging RFUs, relative fluorescence units RT-PCR, reverse transcriptase polymerase chain reaction SAGE, serial analysis of gene expression SAM, significance analysis of microarrays... 

Fig. 3.2 Gene expression analysis with microarrays. cDNA is obtained by reverse transcription of mRNA using oligo(dT) primers. With high-density commercial arrays (A), biotinylated ribonucleotide analogs are incorporated during in vitro transcription of the cDNA. After fragmentation and hybridization, fluorescence detection occurs by streptavidin-mediated binding of fluorophores to biotin. One hybridization is performed per each sample analyzed. Custom-made cDNA arrays...

The ultimate goal of microarray-based expression analysis is to acquire a comprehension of the entire cellular process, in order to exploit and to standardize the multidi-menisional relations between genotype and phenotype. However, an increasingly important parameter, which has not yet been substantially taken into account, is the role of cellular translation. This means that mRNA expression data need to be correlated with the assortment of proteins actually present in the cell. One approach is based on the use of microarrays containing double-stranded DNA probes for the analysis of DNA-protein interaction and, thus, the detection and identification of DNA-binding proteins by means of fluorescence [130] or mass spectrometry analysis [131]. Moreover, substantial efforts are currently under way to develop protein, antibody, or even cell arrays, applicable to the cor-... 

Genome-Wide Analysis of mRNA Polysomal Profiles with Spotted DNA Microarrays... 

In the last few years, DNA microarrays have been utilized to perform simultaneous analysis of the translational status of thousands of mRNAs, thereby enabling a comprehensive view of translation efficiency and regulation. Analyses were performed on mRNAs isolated from various... 

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.

There are numerous protocols for polysomal gradients preparations that differ mainly at the step for harvesting the cells, and the gradient composition and separation times. The protocol presented later was optimized for isolation of polysomal mRNA from the yeast Saccharomyces cerevisiae, yet many steps will be similar to other eukaryotes and the procedure can easily be modified for other organisms. We will use this protocol as a template on which we will indicate and highlight points that are critical for the microarray analysis. Generally, the RNA isolated by this protocol can be used for analysis by DNA microarray, Northern blot, or RT-PCR. 

The methods for genome-wide analysis of ribosomal association are conceptually different from those for analysis of a single mRNA (e.g., northern analysis). While in assays of a single gene by northern analysis it is simple to compare the distribution of an mRNA in different fractions, in a genome-wide assay, the mRNAs are usually compared relative to others within the fraction. Because of that, it is important to analyze by northern analysis several mRNAs that appeared to be affected to different extents, and to compare the relative effects among this group between the microarrays and the northern analysis. 

Levels of RNA (usually specific mRNAs) in a cell can be measured by well-established techniques such as northern blot analysis or by PCR analysis. However, the recent advent of DNA microarray technology has converted the identification and measurement of specific mRNAs (or... 

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. 

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