MicroarraysAn Approach for Analyzing Patterns of Gene Expression

In the past few years, a new approach has emerged designed to assess differences in gene expression between various cell types or the same cells treated in different fashions. This technology, referred to as microarray technology or gene profiling, has the ability to rapidly and reliably scan large numbers of different mRNAs. The principle is simple and is derived from what we already know about RNA and DNA hybridization. mRNA is isolated from a given sample. Then, when cDNA synthesis is initiated the first strand of the cDNA is labeled with the tag. This forms the pool of target sequences.

The next step is to hybridize the labeled cDNA to a microarray. There are many microarrays commercially available, which fall mainly into two classes; those composed of cDNA, and those composed of oligonucleotides. Microarrays of cDNAs are, as the name suggests, a collection of cDNA that have been arranged, or arrayed, on a solid substrate in defined locations. The substrate varies but usually is a nylon membrane or a glass slide. If a very small amount of cDNA is used, the spots of cDNA arrayed on the substrate can be as small as 100-300 ^m in size; it is relatively simple to array as many as 30,000 cDNAs on a single microscope slide (Figure 23-19a). The actual process of arraying the cDNA is usually accomplished using robotics. The cDNAs are most frequently obtained from available cDNA libraries and, in some cases, are PCR products amplified from the cDNA library using primers specific for certain known genes.

The oligonucleotide arrays are usually a collection of oli-gos 20-25 nucleotides long (Figure 23-19b). The advantage of this type of array is that one only needs sequences of genes of interest. No cDNA library is needed. However, the cost of assembling such an array is high, since the oligos have to be made and then spotted onto the filter or glass slide. Another problem with this approach is, depending upon the length of the oligo, there can be a degree of non-specific hybridization that hinders the final analysis of the data. This problem can be avoided by making longer oligos—which further increases the cost. For these reasons, oligo arrays are used most often by large pharmaceutical or biotechnology companies.

Although the source of the targets used for both cDNA and oligo arrays are cDNA, the preparation of the target differs depending upon the microarray. The target preparation for cDNA arrays involves labeling the cDNA with different fluorescent dyes such as Cy3 and Cy5 (Figure 28-19a). Cy3 and Cy5 are cyanine-based dyes that are easily conjugated to nucleic acids and are highly stable and emit less background fluorescence than conventional fluorescent dyes. Suppose you wish to compare two different cell types, or one cell type in two different states of activation. cDNA from one population is prepared using mRNA as a template. First strand synthesis of the mRNA is performed using one nucleotide con jugated to Cy3. Then, using mRNA from the second cell population, cDNA is prepared using a nucleotide conjugated to Cy5. These two populations of cDNA, one marked with Cy3 and the other with Cy5, are hybridized to the microarray. If one of the targets hybridizes to a cDNA on the array, a green (Cy3) or red (Cy5) fluorescence emission is detected. If both hybridize to the cDNA, yellow fluorescence is detected (the combination of the red and green emissions from both dyes). The arrays are analyzed by scanning the array at two different wavelengths to distinguish between the Cy3 and Cy5 signals. Once scanned at two wavelengths, the signals are compared and the signal intensity of each dye is determined and compared. The results are presented as a ratio between the two samples.

In the case of oligo-based microarrays, the usual approach is to label the target cDNA with a biotin-labeled nucleotide during first-strand synthesis of the mRNA. The biotinlabeled cDNA is hybridized to the oligo array and detected by the use of the fluorescent strepavidin (Figure 28-19b). The procedure is then repeated with cDNA from the other cell type and another microarray is used. The resultant microarrays are analyzed by either phosphoimaging or fluorescent-based scanning. This is most commonly accomplished using specialized scanners developed for scanning microarrays.

The difference between this procedure and the cDNA-based array described above is that two microarrays are used. This is possible since the method for producing the oligo-based microarrays is more precise and it is possible to ensure that the same oligo will be present in precisely the same position on two separate microarrays. This is not possible with the technology used to prepare cDNA microarrays. Therefore, both targets must be hybridized to the same array to derive an accurate comparison. There is an advantage to using two microarrays. Quantitation of expression levels is easier when using one labeled target per microarray. When two targets are hybridized to the same array, it is always necessary to "subtract" the fluorescence of one target from the other before it is possible to obtain quantitative data. Since only one target is hybridized to a single oligo microarray, subtraction is not necessary.

The application of microarray technology to immunology is apparent. One could easily ask what is the difference between T cells and B cells. Or what is the difference between an activated T cell and a resting T cell? The list of possible comparisons is immense. To begin to answer some of the interesting immunology questions, Louis Staudt and co-workers at the NIH have developed an array they term "Lymphochip." The Lymphochip is an array that consists of more than 10,000 human genes and is enriched in genes expressed in lymphoid cells. It also includes genes from normal as well as transformed lymphocytes. This particular microarray has provided a great deal of useful information, including a profile of T cells compared to B cells, plasma cells compared to germinal center B cells, and gene expression pattens induced by various signaling pathways. The Lymphochip and other clinical applications of microarrays are described in the Clinical Focus box.

Isolate mRNA

Isolate mRNA

Synthesize labeled cDNA targets, denature

Isolate mRNA

Hybridize to microarray

Bi0tin

Isolate mRNA

cDNA synthesis

Add label

Hybridize to microarray

Hybridize to microarray

Array 1

Measure ratio

Cy5/Cy3

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Measure ratio

Cy5/Cy3

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Measure ratio of label array 1/ array 2

Spot onto Amplify substrate by PCR

Selected cDNA

Array 2

Measure ratio of label array 1/ array 2

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Array 1

Array 2

Spot onto substrate

Microarray preparation

Spot onto Amplify substrate by PCR

Selected cDNA

DNA microarray

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Oligo microarray

FIGURE 23-19

DNA microarray analysis using cDNA microarrays (a) or high-density oligonucleotide microarrays (b). As described in the text, microarray analysis relies on the isolation of RNA from the tissues or cells to be analyzed, the conversion of RNA into cDNA, and the subsequent labeling of DNA during target preparation. The labeled target sequences are hybridized to either a cDNA microarray (a) or an oligo microarray (b).

SUMMARY

■ Inbred mouse strains allow immunologists to work routinely with syngeneic, or genetically identical, animals. With these strains, aspects of the immune response can be studied uncomplicated by unknown variables that could be introduced by genetic differences between animals.

■ In adoptive-transfer experiments, lymphocytes are transferred from one mouse to a syngeneic recipient mouse that has been exposed to a sublethal (or potentially lethal) dose of x-rays. The irradiation inactivates the immune cells of the recipient, so that one can study the response of only the transferred cells.

■ With in vitro cell-culture systems, populations of lymphocytes can be studied under precisely defined conditions. Such systems include primary cultures of lymphoid cells, cloned lymphoid cell lines, and hybrid lymphoid cell lines. Unlike primary cultures, cell lines are immortal and homogeneous.

CLINICAL FOCUS

CLINICAL FOCUS

Microarray Analysis as a Diagnostic Tool for Human Diseases impossible to distinguish visually between B and T cells without molecular analysis. Similarly, it can be quite difficult to distinguish one tumor from another. Two of the best-known acute leukemias are AML, which arises from a myeloid precursor (hence the name, acute myeloid feukemia) and ALL (acute lymphoid feukemia), which arises from lymphoid precursors. Both leukemias are derived from hematopoietic stem cells, but the prognosis and treatment for the two diseases are quite different. Until recently, the two diseases could be diagnosed with some degree of confidence using a combination of surface phenotyping, karotypic analysis, and histochemical analysis, but no single test was conclusive; reliable diagnosis depended upon the expertise of the clinician.

The difference between an ALL diagnosis and an AML diagnosis can mean the difference between life and death. ALL responds best to corticosteroids and chemotherapeutics such as vincristine and methotrexate. AML is usually treated with daunorubicin and cytarabine. The cure rates are dramatically diminished if the less appropriate treatment is delivered due to misdiagnosis.

In 1999, a breakthrough in diagnosis of these two leukemias was achieved using microarray technology. Todd Golub, Eric Lander, and their colleagues isolated

RNA from 38 samples of acute leukemia, labeled the RNA with biotin, and hybridized the biotinylated RNA to commercial high-density microarrays that contained oligonucleotides corresponding to some 6817 human genes. Whenever the biotinlabeled RNA recognized a homologous oligonucleotide, hybridization occurred. Analysis revealed a group of 50 genes that were highly associated with either AML or ALL when compared with control samples. These 50 genes were then used to sample nucleic acid from 34 independent leukemias as well as samples from 24 presumed-normal human bone-marrow or blood samples. The result? A set of markers that clearly classified a tumor as ALL or AML.

The results of the microarray analysis further suggested that the treatments for AML and ALL can be targeted more precisely. For example, an AML expressing genes x, y, and z might respond to one treatment modality better than an AML that expresses a, b, and c. Several pharmaceutical companies have established research groups to evaluate different treatments for tumors based on the tumor's microarray profile. This designer-approach to oncology is expected to produce much more effective treatments of individual tumors, and ultimately, enhanced survival rates.

Microarray analysis is likely to be very useful in the diagnosis of tumors of the immune system. Most notably, a labora tory at the National Institutes of Health (NIH) has developed a specialized DNA microarray containing more than 10,000 human cDNAs that are enriched for genes expressed in lymphocytes. Some of these cDNAs are from genes of known function, others are unknown cDNAs derived from normal or malignantly transformed lymphocyte cDNA libraries. This specialized array is called the "Lymphochip" because the lymphocyte cDNAs are arrayed on a silicon wafer. The group at NIH asked whether they could use the Lymphochip to divide the B-cell leukemia known as diffuse large B-cell lymphoma (DLBCL) into subgroups, an important question because this type of lymphoma has a highly variable clinical course, with some patients responding well to treatment while others respond poorly. Earlier attempts to define subgroups within this group had been unsuccessful. A definition of subgroups within DLBCL could be useful in designing more effective treatments. Using the Lymphochip, the group at NCI identified two genotypically distinct subgroups of DLBCL. One group was comprised of tumors expressing genes characteristic of germinal-center B cells and was called "germinal-center-B-like DLBCL (see Figure). The other group more resembled activated B cells and was termed "activated B-like DLBCL." Significantly, patients with germinal-center-B-like DLBCL had a higher survival rate than those with activated B-like DLBCL. Normally all patients with DLBCL receive multi-agent chemotherapy. Patients who do not respond well to chemotherapy are then considered for bone-marrow transplantation. The data obtained from this study suggests that patients with activated B-like DLBCL will not respond as well to chemotherapy and may be better served

■ Biochemical techniques provide tools for labeling important proteins of the immune system. Labeling antibodies with molecules such as biotin and avidin allows accurate determination of the level of antibody response. Gel elec-trophoresis is a convenient tool for separating and determining the molecular weight of a protein.

■ The ability to identify, clone, and sequence immune-system genes using recombinant DNA techniques has revolutionized the study of all aspects of the immune response. Both cDNA, which is prepared by transcribing mRNA with reverse transcriptase, and genomic DNA can be cloned. Generally, cDNA is cloned using a plasmid vector; the re-

by bone-marrow transplantation shortly after diagnosis. As a direct result of this work, ongoing clinical trials are evaluating how best to treat patients with activated B-like DLBCL.

Gene profiling is not restricted to diagnosis of cancer. This technology pro vides us with a unique opportunity to examine differences between any distinct populations of cells. One can compare which genes are expressed in common or differentially in a naive T cell and a memory T cell. What is the difference between a normal T cell and a T cell dy ing by apoptosis? Comparisons like these will be a rich source of insight into differences in cell populations. The key to using this valuable information will be the development of tools to analyze the vast quantities of data that can be obtained from this new approach.

GC B-Like DLBCL

Activated B-like DLBCL

DLBCL Biopsies

-1—r

GC B-like

-H-HH—1-h

Activated B-like H-h

2 4 6 8 10 Overall Survival (years)

Diffuse large B-cell lymphoma (DLBCL) is at least two distinct diseases. (a) Shown are differences in gene expression between samples taken from patients with either germinal center B-like DLBCL (left, orange) or activated B-like DLBCL (right, blue). Relative expression of the 100 genes (y axis) that discriminate most significantly between the two DLBCL types is depicted over a 16-fold range using the graded color scale at bottom. Note the strikingly different gene expression profiles of the two diseases. (b) Plot of overall DLBCL patient survival following chemotherapy. Gene expression profiles of tumor-biopsy samples allow the assignment of patients to the correct prognostic categories and may aid in the treatment of this complex disease. [Adapted from L. M. Staudt, 2002. Gene expression profiling oflymphoid malignancies. Annu. Rev. Med. 53:303-318.]

combinant DNA containing the gene to be cloned is propagated in E. coli cells. Genomic DNA can be cloned within a bacteriophage vector or a cosmid vector, both of which are propagated in E. coli. Even larger genomic DNA fragments can be cloned within bacteriophage P1 vectors, which can replicate in E. coli, or yeast artificial chromo somes, which can replicate in yeast cells. Polymerase chain reaction (PCR) is a convenient tool for amplifying small quantities of DNA.

■ Transcription of genes is regulated by promoter and enhancer sequences; the activity of these sequences is controlled by DNA-binding proteins. Footprinting and gel-shift analysis can be used to identify DNA-binding proteins and their binding sites within the promoter or enhancer sequence. Promoter activity can be assessed by the CAT assay.

■ Cloned genes can be transfected (transferred) into cultured cells by several methods. Commonly, immune-system genes are transfected into cells that do not normally express the gene of interest. Cloned genes also can be incorporated into the germ-line cells of mouse embryos, yielding transgenic mice, which can transmit the incorporated transgene to their offspring. Expression of a chosen gene can then be studied in a living animal. Knockout mice are transgenics in which a particular target gene has been replaced by a nonfunctional form of the gene, so the gene product is not expressed. The Cre/lox system provides a mechanism that allows tissue-specific expression or deletion of a particular gene.

■ Microarrays are a powerful approach for the examination of tissue-specific gene expression and comparison of gene expression in different cells. It has already begun to revolutionize the study of gene regulation and gene expression.

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