Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 28
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Because of thispriming method, the need of good-quality poly-A RNA is self-explanatory. TheDNA from both samples is subsequently digested by a 4 base-cutter restrictionPrinciple of the subtractive hybridization and amplification of the subtracted products.See text for details.Fig. 5.487885 Genomics by Subtractive Hybridizationenzyme (Rsa I, typically). One of the two libraries is then ligated to specificadapters, before the two preparations are hybridized together. Two successiverounds of hybridization are usually performed. The duration of the hybridization is a crucial aspect of the experiment for the following reason. The hybridization between two complementary strands of DNA is proportional to the concentration (Co) of the strands and the time (t) of hybridization.
The product ofthese two factors (Cot) is specific for each sample of DNA sample. Because ofthe Cot principle, the more abundant transcripts will hybridize faster. As thesubtraction does not affect the concentration (Co) of the cDNAs, adjusting thetime of hybridization (t) is a mechanism of normalization, to ensure that all thetranscripts (rare or abundant) will hybridize relatively equally.
Because of thisnormalization, the chance to find a transcript in the subtracted library after theexperiment is relatively independent of its original abundance. The DNA fragments that do not hybridize correspond to those which are expressed differentlyin the two preparations (Fig. 5.4). These gene products are further amplified byPCR using a single primer pair that recognizes the adapters, and subcloned ina vector system. The plasmid can be used for sequencing, microarrays, Northern blot or Dot blot (Fig. 5.4). The adapters are ligated to one cDNA libraryonly.
For instance, if the adapters are ligated on the cDNA library from an ischemic heart (labeled “tissue of interest” in Fig. 5.4) and if this library is subsequently subtracted from a library from normal myocardium, the experimentwill show the genes that are overexpressed in ischemic myocardium. Reciprocally, the adapters can be ligated to the library from normal heart, to show thegenes that are downregulated in the ischemic territory. A complete hybridization therefore includes a “forward” and a “reverse” library to show both the upregulation and downregulation of genes in one condition compared to another.4.
Sequencing and database query. Although the hybridization in itself can beachieved in less than a week, the identification of the subcloned genes is morelabor-intensive. Nowadays, the sequencing work is greatly facilitated by the useof automated sequencers using a 96-well or 384-well format.
Some investigatorsprefer to verify first the quality of the library by microarrays. The sequencingalso determines the quality of the library by the extent of the different geneproducts found, and by the redundancy of the same products. Finding newproducts by sequencing a library follows an exponential curve. The first batchesof sequences bring many different gene products, then further sequencingshows the same products coming back again and again. It is reasonable to interrupt the sequencing when the last batch of clones does not bring more than10% of new information (for instance, if less than 10 new gene products arefound in a 96-well sequencing plate). Also, if a specific gene is highly regulated(i.e., if the concentration of the transcript between the two conditions testedshows more than a 10-fold difference), this specific product has a high probability to be sequenced more often.5.
Microarrays. Microarrays represent a reliable technique to verify at a glancewhich clones from the library are truly regulated. It consists in spotting all theclones isolated on nylon membranes or glass chips and to hybridize them with5.2 Analyzing Gene Expression by Subtractive Hybridizationlabeled RNA from the different experimental groups tested. The labeling can beradioactive (using 33P isotope instead of 32P for a better resolution) or nonradioactive (using fluorescent dyes or chemiluminescence).
The isotopic methodis limited in its sensitivity. This technique therefore offers some informationabout the magnitude of changes in expression for each gene product. Thesechips can also be hybridized with labeled fragments of the mitochondrial genome. By this approach, it is possible to define which clones correspond to mitochondrial DNA, instead of nuclear-encoded genes.
This strategy may greatlyreduce the number of clones to sequence.6. Validation. Whatever the results of the hybridization and the identity of the isolated clones, a method of validation is always required. It is necessary to rely onmethods that truly quantify the changes in expression of one specific gene ofinterest. One of the most popular methods is the Northern blot, because theprobe is already available in the form of the clone sequenced from the library.The average size of the cDNA fragments obtained during the subtraction (0.6 to1.2 Kb) corresponds to the recommended size of a usual probe for Northernblot. In addition, the tissue localization of the transcript can be validated furtherby in-situ hybridization.
Again, the subcloned DNA fragment can be used as aprobe, although some investigators prefer to use oligo probes. Finally, the quantitative PCR (using specific fluorogenic probes or as a screening technique withthe SybrGreen) will be the preferred choice when analyzing a large number ofsamples, when making a time-course, or when the RNA supply is limited.5.2.2Advantages and DisadvantagesThe main advantage of the subtractive hybridization consists in its unbiased nature. Potentially, all the genes regulated by a specific condition can be found.
Thisrequires a good preparation of RNA and an extensive work of subcloning and sequencing. Ischemia/reperfusion, cardiac hypertrophy and heart failure, in whichchanges in both workload and ventricular function are found, can affect dramatically the expression of genes in the heart and can trigger the expression of genesthat are not normally expressed in myocardium. The subtractive hybridization istherefore an excellent tool for discovery.
It can show “unexpected” gene profiles,or lead to the discovery and characterization of novel genes. The technique istherefore excellent for both the horizontal and vertical approaches describedabove. It starts by a genomic profiling and then, depending on the identity of theclones, can lead to an in-depth analysis of specific products. Another advantage isthat the experiment does not depend on the species studied. The different genome projects have substantially increased the number of sequences available inpublic databases, so that querying sequences from swine, dog or even woodchucktissues is not a problem in practice.The inconvenience of the subtractive hybridization is that, due to its random nature, interesting clones can be “missed”.
The technique is not as comprehensive as aDNA chip (although all the clones collected from a subtraction can be spotted on a89905 Genomics by Subtractive Hybridizationheart-specific, custom-made chip). However, the chance to find a specific gene isproportional to the magnitude of its upregulation in one group compared to theother. Another inconvenience is the requirement of an access to a dedicated sequencing facility. Finally, especially in the heart, it is impossible to avoid the “contamination” of the library by non-nuclear genes from the mitochondrial DNA. Due to thelarge amount of mitochondria in the cardiomyocyte, such contamination can represent 20–50% of the overall library [50, 52]. This contamination can be limited by performing additional rounds of hybridization, at the expense of loosing rarely expressed nuclear transcripts.
The mitochondrial genome includes the genes codingfor the subunits 1 to 6 of NADH dehydrogenase, the subunits 1 to 3 of cytochromeoxidase, the ribosomal RNAs 12S and 16S, the cytochrome b and the subunits 6 and8 of ATP synthase. A last inconvenience is that the subtraction requires to start with2–3 lg of poly-RNA, which may represent about 50 lg of total RNA in the heart.There are some experimental conditions, especially the genomic profiles during development, in which such amount can hardly be obtained.5.3Genomics of Myocardial IschemiaThe discovery that gene expression can be affected by changes in workload [21,32] rapidly led to the investigation of the adaptation of myocardial gene expression in the ischemic heart. Schaper’s laboratory provided pioneering studies on alterations in gene expression in large mammalian models of ischemia/reperfusion.These studies were mainly designed to follow the time-course of changes in genescoding for proto-oncogenes and calcium-handling proteins [53–55].
The proto-oncogenes were chosen because they represent a rapid mechanism of transcriptionaladaptation to stress, whereas calcium-handling genes were studied to correlatetheir expression to the prolonged dysfunction that follows ischemia. Another goalwas to investigate whether repetitive episodes of ischemia/reperfusion inducingpreconditioning are accompanied by a change in gene expression. Several protooncogenes (such as c-fos or junB) showed an increase either during ischemia orreperfusion. Similarly, the Ca2+-ATPase and calsequestrin were found to increaseduring post-ischemic dysfunction. These studies were the first to show that evenshort episodes of ischemia-reperfusion could affect myocardial gene expression.Also, they showed the possibility to investigate myocardial gene expression inlarge mammalian models of ischemic heart disease.
However, the experimentaldesign of this work adopted a “vertical approach”, which precluded the genomicprofiling of these hearts. Because only limited subsets of genes were investigated,the global impact of these changes could hardly be determined.More recently, the technology of microarrays has been used in several rodentmodels to offer a more comprehensive view of the genomic adaptation to ischemia.
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