Technology Insight: Tuning into the Genetic Orchestra Using Microarrays-Limitations of DNA Microarrays in Clinical Practice

DNA Microarray Technology

A DNA microarray is an ordered arrangement of equidistant microscopic DNA spots attached to a solid surface, such as a glass, plastic or silicon chip. Hybridization is performed using corresponding probes that recognize and attach to the solid support; these can be complementary DNAs (cDNAs), oligonucleotides of varying length, or genomic sequences that are either radioactively or fluorescently labeled (Figure 2). An array containing thousands of spots immobilized at predetermined locations can be generated by applying the DNA (e.g. cDNA or oligonucleotides) to the array using pins^[3] or inkjet technology,^[4] or by in situ photolithographic synthesis of oligonucleotides.^[5] For example, if inkjet technology is being used, an adapted color inkjet printer is used to synthesize oligonucleotides: each of the four color reservoirs is filled with a solution containing one of the four nucleotides from which DNA is made. The oligonucleotide sequences for each gene can then be assembled using a simple text file. Thus, the probes of a microarray are made of DNA and these probes are then used to detect the abundance of specific mRNA (transcripts) from the genes that correspond to the sequence of the probes on the array.

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Figure 2.

Gene-expression profiling using microarray analysis. The main steps involved and limitations of microarray analysis will be encountered. Two stages can be distinguished in microarray experiments: a pre-microarray experiment phase (tissue handling) and the microarray experiment phase (labeling of the RNA). In a microarray experiment the glass slide with the labeled DNA of interest plays a key role. As can be seen, a solid surface (in this example a glass microscope slide) contains thousands of spots. Each spot contains a large number of identical DNA fragments. Fluorescently labeled RNA from the samples are subsequently hybridized to the arrays. In this way, the amount of DNA fragments per spot indicates the expression level of a gene. The expression level of thousands of fluorescently labeled genes or spots on one microscope slide can be visualized with a fluorescent scanner. For each gene on the array, the amount of fluorescently labeled RNA bound represents the expression level of that gene in the tumor sample. The intensity of fluorescent signal can be measured and used in the statistical analysis. For each spot, the DNA fragments are derived from one specific gene. Figure courtesy of Dr R Kerkhoven. Abbreviations: DHFR, dihydrofolate reductase; E2F1, E2F transcription factor 1; RB, retinoblastoma; SRC, sarcoma.

The main steps involved and limitations of microarray analysis will be encountered. Two stages can be distinguished in microarray experiments: a pre-microarray experiment phase (tissue handling) and the microarray experiment phase (labeling of the RNA). In a microarray experiment the glass slide with the labeled DNA of interest plays a key role. As can be seen, a solid surface (in this example a glass microscope slide) contains thousands of spots. Each spot contains a large number of identical DNA fragments. Fluorescently labeled RNA from the samples are subsequently hybridized to the arrays. In this way, the amount of DNA fragments per spot indicates the expression level of a gene. The expression level of thousands of fluorescently labeled genes or spots on one microscope slide can be visualized with a fluorescent scanner. For each gene on the array, the amount of fluorescently labeled RNA bound represents the expression level of that gene in the tumor sample. The intensity of fluorescent signal can be measured and used in the statistical analysis. For each spot, the DNA fragments are derived from one specific gene. Figure courtesy of Dr R Kerkhoven. Abbreviations: DHFR, dihydrofolate reductase; E2F1, E2F transcription factor 1; RB, retinoblastoma; SRC, sarcoma.

The concept of DNA or oligonucleotide arrays began in the mid-1980s. The first DNA arrays comprised nylon filters on a glass slide containing cDNA probes; these filters contained a much lower number of probes than the arrays that are currently used and were typically used with radioactively labeled targets. The introduction of pin-based robotic systems made it possible to dispense smaller volumes of DNA (approximately 150 microns) onto a glass slide, thus enabling a higher throughput system that represented one of the first microarrays.^[6,7] A summary of the developments in microarray-based gene-expression profiling is provided in Table 2 .

Microarray analysis of cDNAs spotted onto glass slides was developed at Stanford University and has been used to study the expression levels of large numbers of mRNAs in cell lines and tumors.^[8] In brief, mRNA is isolated from cells and reverse transcribed in the presence of red-fluorescent-labeled nucleotides. The resulting fluorescent cDNA is then mixed with a green-fluorescent-labeled reference cDNA and the mixture is hybridized to the microarray. The reference mRNA is usually prepared from a mixture of cell lines, tumor samples, or normal tissues. Using a fluorescent scanner, the fluorescence level is digitized, and for each cDNA on the microarray the level of gene expression, relative to the reference, is determined and transferred to a linked computer database.

Similar methodology is used for oligonucleotide-based arrays. With this system of microarray analysis, the expression of each gene in a sample is also measured relative to the expression of the same gene in the reference mRNA. The photolithographic synthesis of oligonucleotides is a procedure developed by Affymetrix (Santa Clara, CA); for each gene, several different oligonucleotides are present on the array.^[5] In this technique, the hybridization on the array platform is carried out using RNA from the sample to be analyzed, without the use of a reference RNA; instead, oligonucleotides containing one mismatch in their sequence are used to correct for background hybridization. An array containing 25,000 probes can provide information on the expression of all genes present in the genome. For many genes, multiple splice variants have been identified, and for sufficient array detection an increasing numbers of probes are being developed.

Gene-expression profiles can provide an enormous amount of information on cell function; however, it is important to realize that these profiles can only be used to interpret cellular changes that affect mRNA synthesis. After translation of mRNA into protein, many secondary protein modifications also play an important role in cell regulatory processes. Therefore, high-throughput analysis of proteins ('proteomics') will also contribute greatly to cancer research, an issue that was recently reviewed by Gulmann et al.^[9] Proteomic experiments can be tedious and have a lower throughput than RNA-based experiments;^[10] however, recent advances in proteomics have greatly increased the throughput of proteomic experiments.

Recently, small RNA molecules that do not code for proteins, such as microRNA, RNA interference, small interfering RNA (siRNA), and small modulatory RNA, have been discovered; these small RNAs have an important role in gene regulation.^[11] Genes that regulate the expression of molecules such as microRNA are found in the non-coding regions (or introns) of the genome. Although these RNAs do not code for proteins, they can control gene expression at the post-transcriptional level by degrading or repressing mRNA,^[11] thus influencing critical functions and biological processes.^[12] Although microRNA consists of only 1-5% of human genes (which equates to about 1,000 microRNA genes), each microRNA might regulate as many as 200 genes, which implies that over one-third of the genes that encode for proteins are regulated by microRNAs.^[11,13] MicroRNA expression profiles can be used to classify human cancers^[12] because they reflect the developmental lineage and differentiation state of the tumors. Compared with normal tissue, microRNAs in tumors are generally downregulated and could thereby provide promising insight into tumor development and recurrence. For example, microRNA profiles are better predictors of metastatic origin for carcinomas of unknown primary (CUPs) than are conventional gene-expression signatures using mRNA.^[14]

Nat Clin Pract Oncol © 2006  Nature Publishing Group

Cite this: Technology Insight: Tuning into the Genetic Orchestra Using Microarrays-Limitations of DNA Microarrays in Clinical Practice - Medscape - Sep 28, 2006.