Taking The Measure Of The Message

Date: November 23, 1998Product Comparison Getting a measure of even a single RNA species has never been easy. Every few years, a new technique comes along--Northerns, ribonuclease protection assays, RT-PCR--that makes the task only incrementally easier. But a quantum leap in technology took place just a few years ago with the introduction of cDNA arrays, and suddenly not just one but hundreds to thousands of target nucleic acids can be analyzed simultaneously and precisely. Since then, array technology has become a growth industry as new tools for making, probing, imaging, and analyzing arrays are being introduced almost daily. Old-Fashioned Way: One Message at a Time Scan of Incyte's UniGEM Microarray. Image courtesy of Genome Syatems. Quantitating mRNAs has been fraught with difficulties due to the lability of RNA and the vanishingly small amounts typically present in cells. Consequently, techniques that are robust and sensitive have been in demand. High on the list in sensitivity is RT-PCR, although a new assay based on structure- specific endonucleases has been described that rivals RT-PCR (L. De Francesco, The Scientist, 12 (21):16, October 26, 1998). However, RT-PCR, while sensitive, requires some machinations to render it quantitative. A somewhat less arduous method for quantitating mRNA is the ribonuclease protection assay (RPA). While this method falls a little short in sensitivity when compared to RT-PCR, the assay is simpler to run and is more forgiving when it comes to sample preparation. Both RT-PCR and RPA rely on sequence-specific primers or probes and look at individual or, at best, small collections of messages at once. But if that is what you want or need, these techniques can get you there. A number of kits profiled in this article are available to assist you. With the introduction of GeneChip® probe arrays by Affymetrix in 1996, a paradigm shift took place in the field of expression analysis. With this technological breakthrough, it became possible to monitor the expression of sequences in toto rather than in isolation, to view it as part of an integrated process rather than as individual events. The beauty of this has not been lost on researchers and companies as both groups make their way into this burgeoning field. The Affymetrix chip has retained its position at the top of the line in array technology, both with respect to coverage of the genome (Affymetrix expression chips contain probes for every known gene in the human and yeast genomes) and expense (the instrument system used with silica chips costs over a hundred thousand dollars). This technology is obviously not for the average researcher. Accordingly, a number of alternatives have appeared to satisfy researchers at all budget levels. While the Affymetrix silica-based chips have set the stage for this new technology, nucleic acid arrays are being made on other matrices as well. Relying on familiar technologies, some companies offer arrays on membranes, which have the virtue of not requiring specialized equipment for their use. In fact, with a little ingenuity, it is possible to make and analyze your own arrays using existing technology. However, once you reach three-digit numbers, the logistics of working with such large collections of clones may be daunting for some labs. So for the less adventurous, membrane arrays are available from several companies in different sizes and shapes. CLONTECH offers cDNA expression arrays on nylon membranes in which the genes are organized in sectors according to the cellular pathway in which they function, such as apoptosis, oncogenesis, or cell-cycle regulation. The CLONTECH Atlas cDNA arrays contain 588 human, mouse, or rat cDNAs, spotted in duplicate, with each spot containing 10 ng of a 200--600 base pair PCR fragment. According to the company, these filters can detect two to three fold differences in expression levels, and are sensitive enough to detect rare (0.001% abundance level) mRNAs. CLONTECH also offers 13 application- targeted arrays--neurobiology, hematology, immunology, for example--with between 100 and 400 clones per array. In the future, CLONTECH's Atlas cDNA clones will be available on microscope slides as well as membranes. Jointly with Molecular Dynamics, CLONTECH will be developing microarrays and the software and chemistry tools required for their use. Display Systems Biotech offers membranes spotted with a set of fragments from a restriction fragment differential display (RFDD-PCR) analysis, which can be used to look for new family members of expressed genes. At present, membranes for human adult and fetal brain are available. The company also offers a kit designed for producing cDNA probes for analyzing its arrays. Genome Systems offers membranes with over 18,000 nonredundant clones taken from the IMAGE consortium collection of expressed sequence tags (EST). While each clone is not completely defined in terms of function, its sequence is known. Genome Systems constructs arrays by spotting individual bacterial colonies on filters and processing the filter for hybridization. The company has data indicating the reproducibility of its process for generating arrays. Research Genetics is up to its fourth release of Human GeneFilters™ totaling over 20,000 unique genes spotted onto positively charged nylon membranes (5 x 7 cm). Each spot contains 5 ng of insert DNA corresponding to an IMAGE clone averaging 1.5 Kb and bearing the 3' UTR. These clones represent single gene clusters reported in the Unigene Database provided by the NCBI. Each GeneFilters(tm) release contains over 5,100 non-redundant clones and the company's goal is to reach at least 40,000 different human clusters. Research Genetics provides also membranes containing all the yeast genes and is working on mouse and rat cDNA arrays. Surpassing even the largest membrane array in numbers of targets, if not in ease of use, are chips paradoxically referred to as micro-arrays. ("Micro" refers to the physical dimensions of the array, not the number of clones contained therein.) Affymetrix and Incyte offer high-density chips with arrays of thousands and thousands of complementary oligos. The Affymetrix GeneChip product line includes sets of expression arrays for human, murine, and yeast genomes, each sporting over 6,000 sequences. Affymetrix manufactures workstations to carry out the hybridization and washing reactions and has partnered with Hewlett-Packard in the production of a GeneArray™ Scanner. Affymetrix's GeneChips system can now be purchased through Amersham Pharmacia Biotech. Genome Systems has also entered the microarray market with glass chips made by Incyte. A set of four UniGem chips, each containing 10,000 individual amplified cDNA targets, chemically bonded to glass, are being made available to researchers through a service arrangement. (Incyte also has chips with proprietary Incyte sequences, available only to Incyte subscribers.) The hybridization as well as the analysis is done in-house. Genometrix Inc. offers expression microarrays either in 96-well format or on microscope slides for analyzing cancer-related gene expression and mouse gene expression and is working on an array for risk/toxicity assessment. A proprietary technology developed at Baylor College of Medicine is used to generate the Universal Arrays™, which are formed in a self-assembling process based on the charge density of surface films made with synthetic peptides. Genometrix also supplies the BioScanner™ for high-density imaging and GeneView™ software for analysis. Multiplex Quantitative RT-PCR with 18S rRNA as an Internal Control. RT-PCR reactions on various tissues total RNA using A) primers for clathrin, B) primers for clathrin and 18S, or C) primers for clathrin, 18S and 18S Competimers. Figure courtesy of Ambion. While the convenience of pre-fabricated arrays will appeal to some, there are always people, (and a high proportion of these types is found among the ranks of scientists) who prefer to do things for themselves. For those people, robotic devices can either be fashioned or purchased that will draw up collections of samples from a microplate and spot small volumes onto slides. Already there are half a dozen such devices, available from BioRobotics, While macroarrays do not offer any new challenges when it comes to data collection, high-density microarrays do. A typical expression experiment requires using two different fluorescent labels, with the ratio of the two providing a measure of differential expression. In order to be up to the task, imagers must therefore have at least two different channels or wavelength capabilities, as well as be able to scan at high resolution. Among the available imager Packard Instrument's BioChip Imager™ and others that are CCD camera based systems (Genomic Solutions' GeneTac™ and Imaging Research's Array Vision). For researchers wishing to participate in the development process itself, Amersham Pharmacia Biotech/Molecular Dynamics has the Microarray Technology Access Program that allows researchers to make and scan microarrays in their laboratories. This emerging technology is being made to researchers for a fee. So where do you go from there? Once you have identified cDNAs that show an interesting expression pattern, what comes next? With some arrays it is possible to purchase clones of interest for further experimentation. Often the first item of business is confirming that the particular clone you now have in your hand shows the expression pattern so indicated by the array, which brings us back to where we started--quantitating individual mRNA species. RT-PCR, the most sensitive of the available tools, is by its very nature difficult to quantitate; due to the exponential nature of the reaction, even small variations in conditions can lead to large differences in product. In addition, for a variety of reasons (primer exhaustion, loss of activity of the enzyme, and so on), the reaction can plateau, which requires knowledge of the kinetics of each reaction before quantitation can be trusted. To overcome these difficulties, two kinds of PCR reactions have been developed: quantitative (or relative) RT-PCR and competitive PCR. Relative, quantitative RT-PCR uses an internal standard to monitor each reaction and allow comparisons between different reactions to be made. To do this, a second set of primers is incorporated into the reaction for an invariant, housekeeping message. The difficulty here is matching the level of the internal message to that of the target so that one reaction doesn't dominate. The QuantumRNA™ Kit from Ambion uses a patent pending technology for manipulating the amplification efficiency of its internal standard (in this case 18S RNA-certainly ubiquitous) without affecting the efficiency of the PCR reaction with the target species. US Biological employs a similar approach in its Quantitative Reverse Transcriptase Kit, in which a G6PD template sporting a point mutation is added to the sample, which competes with endogenous G6PD, thereby providing an internal standard for the RT-PCR reaction. CLONTECH offers primers for a variety of housekeeping genes that vary in size and have different expression patterns, such that some are more suitable for a particular cell or tissue type. Finally, purely exogenous standards (template plus primer) may be added to the reaction, which can give a signal against which the unknown can be compared, providing a relative estimate of the amount of a target species. The synthetic (control) messages present in Ambion's QuantumRNA Kit and in US Biological's kit can be used in this way, rather than as competitors. Competitive RT-PCR should be that--a competition between a known amount of a template and an unknown target. This method avoids difficulties created by differences in the efficiency of the PCR reaction itself with different template/primer sets. A competitive template binds the same primers but has been altered in some way (small deletions, point mutation) to provide a product that is distinguishable from the target itself. The kits with the widest application for competitive PCR or RT-PCR are PanVera's Competitive DNA Construction Kit and Competitive RNA Construction Kit. These kits direct the production of competitive templates for use with any target by providing a scheme for attaching primer sets to a lambda template at various positions depending on the size of the PCR fragment desired. While these kits provide a general method for producing a competitive template (RNA or DNA), the template is not identical to the target, except at its ends (the primers themselves) and hence, some there still might be differences in PCR efficiency. Next in line are kits that provide competitive templates--mostly deletion bearing but otherwise homologous templates--for particular mRNA species that can be used for quantitating only that message species. Competitive kits come in two varieties: those with competitive DNA templates (Biosource International, Cayman Chemical, and Maxim) and those with competitive RNA templates (Ambion). Cayman Chemical has kits for nitrous oxide and cyclo-oxygenase (mouse and human); Ambion, BioSource International (the only non-gel based assay, BioSource Kits are microplate assays, and Maxim offer roughly a dozen different kits, mostly for various cytokines. For the serious student of quantitative PCR, two systems are available (Perkin-Elmer's TaqMan and Idaho Technology's Light Cycler, now available from Roche Molecular Biochemicals®)that are fully integrated PCR amplification and detection systems. These thermal cyclers are closed-tube systems with built-in fluorimeters that quantitate PCR in real time using fluorogenic primers. Fancy and expensive, these systems can't be beaten in terms of speed and accuracy. CLONTECH's Atlas Rat cDNA Expression Array As mentioned earlier, RPA is easier to quantitate than RT-PCR but may not have the sensitivity required to detect the rarest mRNA species. In the ribonuclease protection assay, template-specific, radioactive probes are hybridized to solutions containing mRNA, forming ribonuclease-resistant duplexes. Following hybridization, the samples are treated with ribonuclease to remove unreacted probe and message, and the resultant resistant duplexes are run out on gels, visualized by autoradiography and quantitated usually by phosphor screen imaging. This technique offers advantages both in the efficiency of the reaction, since the hybridization is done in solution, (which is more efficient than membrane-bound hybridizations), and in that it is fairly forgiving of the quality of the preparation used. And as techniques for making and detecting hot probes improves, the sensitivity may soon rival that of RT-PCR. Ambion offers a number of kits for RPA: the basic RPA and two variations that allow for multiple probes and multiple targets to be used simultaneously; HySpeed™ RPA, which reduces the time for the hybridization to a few hours (proprietary buffer); Direct Protect™ Lysate RPA, which allows for RPA to be conducted directly on GITC lysates; and Multi-NPA™, which looks at multiple species at once. PharMingen offers a series of kits with a collection of probes for detecting up to 11 mRNAs in a single sample. The collections include cytokines and hemokines, cytokine and chemokine receptors, cell surface antigens, apoptosis-associated molecules, cell-cycle regulators, oncogenes, and others. This is just the beginning. "Chips," in the words of one company official, "are not a fad, they are here to stay." So look for more and different chips and arrays in the future, as well as more products to enable access to this emerging technology. Absent from this profile on cDNA quantitation are a number of other more open-ended technologies for identifying expressed genes and quantifying message species, such as differential display, amplified fragment length polymorphism (AFLP), or SAGE. These techniques, some of which may spawn growth industries themselves, have been discussed previously in these pages (J. Kling, The Scientist, 12 (17); 9, August 31, 1998; L. DeFrancesco, The Scientist, 12 (9); 19, April 27, 1998). Product Comparison For additional information on this product, please call CompuCyte at 800-840-1303 or visit the company web site: www.compucyte.com. Author Laura De Francesco can be reached at ldefrancesco@the-scientist.com.

Taking The Measure Of The Message

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