Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 23
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Worse still, the artifacts or inappropriate interpretation of the results may lead to blind-ended follow-up experiments that should never havebeen.3. Inadequate quality control of the chipsets. Even though chip production is nowmostly automated and generally of high quality, there are still many opportunities for errors from improper spotting to the wrong sequence selection. Thistype of error can become magnified when it involves a large number of thegenes from multiple experiments.4.
Inability to detect low abundance gene transcripts. The abundance of a gene transcript does not relate directly to its functional importance. Indeed, small criticalregulatory messages may be present in low quantity. Chip manufacturers such asAffymetrix and Incyte have increased their sensitivity to approximately one gene/100,000 of genes in TRNA population [12]. This level of sensitivity unfortunatelyis still poorer than that of classical methods such as Northern blot.
Therefore, themicroarray still can miss rare gene expression events, which may be important inthe overall pathophysiology.5. Artifacts from different sources during the various processing steps, includingsample preparation, RNA extraction, labeling, hybridization, cDNA spotting orchip scanning, pose additional problem. However, obsessive care during theprocessing and multiple replications of the experiments can reduce these problems to a minimum.6. A surfeit of data can only be analyzed by dedicated data mining tools or bioinformatics analysis packages, along with the experts in the field with a biological insight.
At the present time, confirmation of microarray results with classical techniques, such as RT-PCR, or real time or other quantitative PCR techniques is still necessary. However, this most likely will no longer be necessarywhen the techniques mature further and results become more reproducible.7. Expectation of immediate functional insight. Little functional information is typically derived from the first analysis of the expression arrays. Functional insights can only be gained through further evaluation of the known literature,the context of a gene being activated or silenced, and the partners with whichthe gene putatively interacts.4.6Experimental DesignThe structure of the microarray data, the appropriate types of analysis, and thequality of the results are influenced by the experimental design.
Careful forethought and planning is needed to design a successful experiment. Because microarray systems are so sensitive any small changes in sample-to-sample treatment, RNA extraction, sample handling, probe labeling, and other steps in theprocessing are likely to affect the results. Every effort must therefore be taken in65664 cDNA Microarrays in Heart Failure Researchexperimental design, such that variations in the data are due to conditions underinvestigation and are not due to artifacts.Whilst it is recognized that microarrays are relatively expensive, it is still important to incorporate a finite number of biological replicates in each experiment toensure ultimate statistical robustness.
This is particularly important in a systemthat has much inherent biological variation. It is also needed to identify low abundance transcripts and/or small changes in expression levels. The most importantpoints to consider when designing an experiment are:· what is the question that we are trying to answer using this technique,· gene expression level of what conditions are compared,· are there any known expression level for genes in these condition which can beused as a reference marker to confirm the fidelity of our result,· identify the areas which can be the source of variation and try to eliminatethem as much as possible, and finally· what is the maximum of the replicate that is possible to use to allow us the useof statistical method.The simplest microarray experimental design is to determine the changes in geneexpression patterns across a single factor of interest, e.g.
temporal frame shifts,genetic manipulation of a single gene, or effects of drug treatments. However, experiments can now also be designed in a multi-factorial fashion to assess their interactions in one set of microarray experiments. Statistical methods are now available to determine the appropriate number of replicates, or to assist the researcherto design appropriately powered experiments [13].4.7Tissue Preparation and PreservationThis is arguably the most important step for RNA stabilization. Reliable results depend the integrity of RNA. Immediate RNA stabilization in the heart (or any biological) sample is necessary, because changes in gene-expression pattern occur rapidlydue to specific and non-specific RNA degradation.
There are several methods to stabilize the RNA. The simplest and most widely used is snap freezing of the tissue inliquid nitrogen within minutes of removal. The sample then must be preserved at avery low temperature in a nuclease free environment to ensure to sterility and freedom from nuclease contamination, specifically RNAase contamination. Alternatively, samples can be submerged in the RNA stabilizing reagent (e.g.
RNAlater,QIAGEN) immediately after harvesting and stored up to 4 weeks at 8 8C or archivedat –20 8C or –80 8C. This RNA stabilizing reagent is specific for animal tissues andcan be used for cell-culture and white blood cells, but not for whole blood, plasma, orserum. The advantage of this method over snap freezing is the convenience in cutting and handling of the tissue, that can be transported and stored at close to ambient temperatures.4.9 RNA Amplification4.8RNA IsolationThis is a crucial step for preparing high quality RNA free of RNase contamination.Glassware must be treated with DEPC-H2O (0.1% DEPC in H2O) before use.
Boththe quality and quantity of RNA yield improves if samples are handled with care toavoid RNAase contaminats. Heart tissue is considered a challenging tissue for theisolation of RNA because it is fibrious and thus difficult to homogenize and processfor RNA isolation. Several methods are available to purify RNA (either total ormRNA) from the heart tissue. In addition to several commercially available kits,the major method of RNA isolation uses a Guanidine containing reagent, such asTRIZOL (Gibco). Frozen, or RNAlater stabilized tissue samples are disrupted mechanically in a reagent or under liquid Nitrogen and homogenized. At this point,an additional step is necessary to eliminate the fibrous tissues from the sample before proceeding to phase separation with Chloroform.
After phase separation, theTRNA is precipitated using Isopropanol and is washed in 70 ethanol (DEPCH2O). It is estimated that approximately 0.1–1 pg of TRNA is present in a singlecell [14]. TRNA may vary with sample condition, viability of cells, functional status, and phenotype of the cells. The concentration of RNA can be determined bymeasuring the OD at 260 nm (A260) in 10mM Tris.Cl, pH 7.5. TRNA with A260/A280 ratio of 1.9–2.1 is used for microarray experiments.
Integrity and size distribution of TRNA must be checked using denaturing agarose gel electrophoresis. In caseof small samples, such as those obtained by laser capture microdissection or biopsies, spectrophotometer reading can be omitted because too low RNA concentrationmay produce false negative OD values. The best method of RNA characterization inthese cases is the RNA Bioanalyzer (Agilent). The system permits rapid screening ofRNA samples with each disposable RNA chip to determine the concentration andpurity/integrity of 12 RNA samples with a total analysis time of 30 minutes. PurifiedRNA may be stored at –80 8C in water, with no detectable degradation after one year.4.9RNA AmplificationResults of the human genome project [15–17] have laid the foundation for the microarray gene expression profiling [18, 19].
However, broader utilization of microarray methods is limited by the amount of RNA required (typically 10 lg ofTRNA or 2 lg of poly (A) RNA) [12]. This is especially a problem with limitedsamples, such as endomyocardial biopsies and laser capture microdissection(LCM) can be obtained. An important frontier in the development of microarrayfor expression profiling involves reduction of the required amount of RNA. Methods aiming at intensifying the fluorescence signal have resulted in an improvement [20]. Significant increase in detection level can be achieved by amplifyingpoly(A) RNA or cDNA [21, 22]. There are two primary approaches which can beemployed to overcome RNA limitations. One is PCR-based amplification and re-67684 cDNA Microarrays in Heart Failure Researchproducible yield, but the relative abundance of the cDNA products is not well correlated with the starting mRNA level.
The second approach avoids PCR and utilizes one or more rounds of the robust linear amplification based on cDNA synthesis and a template-directed in vitro transcription reaction [14]. This is a recommended method for amplifying the low abundance mRNA or even gene expression profiling of a single cell by orders of magnitudes from nanograms (1–50 ng)of TRNA or poly(A) RNA in one or two round(s) of amplification(s).
This methodcombines a reverse transcription step with an oligo (dT) primer that contains a T7RNA polymerase promoter. The first-strand cDNA is then used for synthesis ofsecond-strand DNA by DNA polymerase, DNA ligase, and RNaseH. The resultingdouble-strand cDNA functions as a template for in vitro transcription step (one ortwo rounds) which results in a linear amplification of RNA.
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