Table of contents Letter from the Editor . . . . . . . . . . . . . . . . . . . . . . .5 Index of Experts . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Q1: Which sample collection and storage techniques yield optimal RNA? . . . . . . . . . . .7 Q2: What techniques do you use to assess and isolate high-quality RNA? . . . . . . . . . . . .9 Q3: How do you establish controls for heterogeneous or difficult samples? . . . . . . . .10 Q4: How do you improve hybridization specificity and sensitivity? . . . . . . . . . . . . .11 Q5: What tips do you have for clean-up and handling of arrays? . . . . . . . . . . . . . . . .12 List of Resources . . . . . . . . . . . . . . . . . . . . . . . . . .14 Genome Technology Microarray Sample Prep Tech Guide 3 Biomek 3000 with the ArrayPLEX application Now available on the small-footprint Biomek® 3000, the ArrayPLEX application provides GeneChip customers with: • Streamlined workflow with validated methods – from 1 to 96 samples processed in a single run • Superior Agencourt® mag bead purification technology yielding high-quality and high-quantity cRNA • Automation that is flexible and powerful, with proven field performance Biomek FX with the ArrayPlex application For more information about ArrayPLEX, visit www.beckmancoulter.com/arrayplex *GeneChip and Premier are trademarks of Affymetrix. © 2006 Beckman Coulter, Inc. Letter from the editor Welcome to the latest installment in Genome Technology’s reference guide series. In this second issue, we are pleased to present expert insights on optimizing sample prep for microarray experiments. Sample prep can make or break your microarray experiment. Whether your samples come from the clinic or the bench, the tips compiled in the following pages will help you maximize the quality of your arrays. Since making their debut, microarrays have gained popularity at a remarkable pace. Commercial chips have found their way into labs everywhere, luring investigators with the promise of generating reliable data on thousands of genes at once. The tool will no doubt continue to reach new users as the costs involved in setting up and running a microarray experiment keep dropping, while dedicated core facilities spring up in both universities and industry. Genome Technology erie-genome-half-h.indd 1 Microarray technology may be widely available, but important issues remain. From sample selection to data analysis across platforms, groups are working to agree on a number of quality control guidelines for all steps in the process. Only in this way can microarrays go from standard tool to standardized technology. One of the most critical steps in the process is sample preparation. Accurate and meaningful data is highly dependent on the quality of isolated nucleic acid or protein samples, and these, in turn, are contingent on correct acquisition, extraction, and purification procedures. Learning how to master correct handling of samples takes experience, which can be costly to attain (in both reagents and time). The microarray experts on our panel offer detailed advice on issues in sample prep. Keep this guide on hand for quick answers to common problems concerning sample collection, isolation, establishing controls, and more. — Jennifer Crebs Microarray Sample Prep Tech Guide 5 12/9/05 10:43:20 AM Index of experts Genome Technology would like to thank the following contributors for taking the time to respond to the questions in this tech guide: Shoshi Kikuchi Head of the Laboratory of Gene Expression Department of Molecular Genetics National Institute of Agrobiological Sciences, Japan Philippe Demougin Biozentrum University of Basel André Ponton Director, Microarray Laboratory McGill University and Genome Quebec Innovation Centre Gary Hardiman Director, BioMedical Genomics Microarray Facility Assistant Professor, Department of Medicine University of California, San Diego Scott Tenenbaum Assistant Professor, University of Albany-SUNY GEN*NY*SIS Center for Excellence in Cancer Genomics Five minutes a day keeps you ahead of the helix. Visit 2, 3, 4 times a day for concise, authoritative news reports on the business and technology of genomics, updated continuously throughout the business day. www.GenomeWeb.com 6 Microarray Sample Prep Tech Guide Genome Technology Which sample collection and storage techniques yield optimal RNA? contact with RNA. So in this case tissues must be The key parameter for yielding optimal total RNA homogenized (in a solution inhibiting RNase activity) quality is to immobilize tissues as fast as possible very rapidly going from a frozen stage (transport after sampling. Proceeding fast is crucial because it samples on dry ice) to a completely homogenized doesn’t let time for RNA level of expression to stage within seconds. change because of technical stress, and more — Philippe Demougin importantly, RNases won’t have time to get in contact with RNA molecules. I have found that both solid Tissues can be fetched into liquid nitrogen or in RNAlater “Solid phase methods work phase and chaotropic salt methods give adequate results. (Ambion, Cat# 7020). Either very well in that they The advantage with chaotropic way is good but RNAlater requires the samples to be small generate very clean RNA, but salt methods is that once released from the cells or so that the solution can get into there is greater risk of tissues, the RNA is less prone to the tissue and protect RNA. initial degradation due to the RNAlater is preferred over liquid degradation.” inhibitory effects of guanadium nitrogen especially in two — Gary Hardiman and phenol. These can later be cases: in surgery room where a problem as they can interfere the use of liquid nitrogen is with the reverse transcription commonly prohibited and in reaction by inhibiting the activity of the reverse case further dissection steps are required for transcriptase enzyme. isolating a tiny part of tissue (embryo Often I see RNA that is structurally intact but microdissection for instance). cannot be converted into high-quality target material Tissues are left in RNAlater overnight at 4°C, due to the presence of contaminants. Ethanol then stored at -20°C or -70°C. If snap-frozen in precipitation or clean-up with a solid phase method liquid nitrogen, they are stored at -70°C following is required. Solid phase methods work very well in snap freezing. Proceeding to tissue isolation, the use that they generate very clean RNA, but there is of RNAlater allows more flexibility. Since the solution greater risk of degradation. This means that an penetrated the tissue before storage, samples can be RNase inhibitor must be used. Additionally, the yields thawed on ice and tissue homogenization is of RNA are generally lower. performed with no rush. — Gary Hardiman For tissues stored at -70°C after snap freezing in liquid nitrogen, the situation is more critical: even We are preparing total RNA from plant tissue. For the partial or short thawing can result in dramatic RNA microarray analysis and degradation because the tissues are destructurized (continued on page 13) RNA preparation, to by the temperature shock and RNases will be in Genome Technology Microarray Sample Prep Tech Guide 7 What techniques do you use to assess and isolate high-quality RNA? Whatever the tissue from which RNA is isolated, we systematically pass total RNA over a silica-based column (e.g. Qiagen RNeasy) in order to clean it up from genomic DNA, salts, phenol… We routinely used a Qiagen TissueLyser apparatus (Cat# 85220) for homogenizing the tissues. Tissue is [shaken] at high frequency in a tube in the presence of a single 5mm metal bead. The protocol applied depends on how difficult it is to disrupt the tissue and open up the cells: if the moderate lysis power of the lysis solution (RLT Buffer) provided with the RNeasy kit is sufficient, homogenate is directly passed over the silica columns. This is typically the case for cell culture or soft tissues. But a phenol-based chemical (e.g. TRIzol) is required for most tissues either because they are tougher to break up, richer in proteins, or richer in lipids. One can adjust the time and frequency of homogenization. 25Hz for two minutes is a safe starting point. One should always homogenize the minimum time necessary for a complete homogenization. — Philippe Demougin High-quality RNA can be isolated using either chaotropic salt and solid phase methods, or a combination of the two. The RNA can be assessed by spectrometry and gel electrophoresis if quantities permit. The ratio of the absorbance readings at 260 and 280 should be between 1.8 and 2. Typically the ratio of high to low molecular weight ribosomal RNA should be about 2. DEPC-treated water should be avoided for resuspending RNA as this can inhibit enzymatic Genome Technology reactions. Additionally, if there is a need to concentrate the RNA, this should be done by salt precipitation or desiccation with a low heat setting. Once RNA has been dried to completion, it can be very hard or impossible to re-suspend. Tissue provides much more of a challenge for RNA extraction than cell lines. Typically, the material needs to be well homogenized prior to extraction. When using a chaotropic salt method, it is critical to avoid organic phase contamination of the RNA supernatant. After centrifugation, one should avoid taking material near the aqueous organic interphase as this is a source of genomic DNA contamination. Agilent Bioanalyzer analysis is very useful in assessing the integrity of total RNA. The 28 and 18S ribosomal RNA species should be present as strong peaks. There should be an absence of low molecular weight products (indicating degradation) and high molecular weight contaminating genomic DNA. — Gary Hardiman [In order to homogenize plant tissue prior to RNA extraction] we are using Multi-Beads Shocker (Yasui Kikai). This product is the most important tool for the preparation of RNA or DNA from hard plant tissues. Many researchers in the plant field out[side] of Japan want to purchase this tool, but [it] is only purchasable in Japan. — Shoshi Kikuchi We received all kind of RNA from different tissues and species. In general TRIzol extraction followed by Qiagen column clean-up give good results. — André Ponton Microarray Sample Prep Tech Guide 9 How do you establish controls for heterogeneous or difficult samples? wavelengths) using a NanoDrop spectrophotometer A mini-electrophoresis system such as the Agilent will reveal the purity of the RNA and the presence of Bioanalyzer or the Bio-Rad Experion allows fast DNA, proteins, or carbohydrates. The integrity of the analysis of RNA samples. Most importantly, they RNA can be assessed using an Agilent Bioanalyzer allow a precise evaluation of their quality. The and compared to control RNA samples. integrity of 28S and 18S ribosomal RNA is used to — Gary Hardiman assess the quality of the total RNA sample. The sharper the peaks, the better the quality. A unique We are measuring the amount of RNA by [using] feature of the Bioanalyzer is the scoring (from 0 to NanoDrop OD measurement equipment and by the 10) of the total RNA quality. For the first time Bioanalyzer system, [by which] degradation of RNA researchers have a tool for assessing the RNA quality samples is monitored. in an objective manner. RNA quality is very much — Shoshi Kikuchi dependent of the tissue, some tissues being naturally richer in RNases than others. By “You have to do a lot of We use the same standard experience we simply know that most people use. what pattern and score to get repeats … our standard is controls Using bacterial spikes and for various tissues. For difficult three biological replicates, things like that, one can samples, measures are taken for one sample from the shortening the times, increasing each technically replicated average next by gauging off of those the homogenization force if twice.” — Scott Tenenbaum types of controls. But I find necessary, working on ice, and those to be only partly useful. working faster. Getting things They tend to control for certain aspects of the organized in order to work faster helps a lot. technique but not the whole method. For instance, A large amount of contaminated genomic DNA the bacterial spikes will control for labeling and in an RNA sample can lead to unexpected migration hybridization issues, but not really for earlier RNA profiles and misleading interpretation. quality. There’s no way to control for that, because — Philippe Demougin you’re adding the control a step after the process occurred. The most difficult samples I have found for You have to do a lot of repeats. I’m funded microarray experimentation are those derived from through the ENCODE project, which is part of the laser capture experiments, where the yields are low NIH and the National Human Genome Research and there may be contaminating genomic DNA Institute, and our standard is three biological present. The extracted RNA should be compared to a replicates, each of which is technically replicated control — for example, the Stratagene or Clontech twice. If you do literally six reference RNA. If sufficient RNA is available, optical repeats on something, an (continued on page 13) density readings at 260nm and 280nm (and other 10 Microarray Sample Prep Tech Guide Genome Technology How do you improve hybridization specificity and sensitivity? We hybridized only Affymetrix arrays. Procedures are very much standardized, and we stick to the default recommendations. Reliable arrays and [automated] procedures being the key advantages of the GeneChips versus spotted arrays. — Philippe Demougin For the various commercial catalog arrays, I usually adhere to standard operating protocols, as much of this has been worked out and optimized by the array manufacturer — deviating from the SOPs can mean you are on your own if the experiment fails. For homemade or boutique arrays where optimization is usually required, several components can be adjusted to improve hybridization specificity and sensitivity. Firstly, good oligonucleotide design is key. The investigator needs to ensure that the oligos are specific to the mRNA of interest, and design probes that do not contain fold-back loops. Hybridization conditions are optimized depending on the slide surface employed; sometimes a pre-hybridization step may improve the specificity. The composition of hybridization buffers is important. I favor formamidebased buffers as you can reduce the temperature and maintain hybridization specificity. One of the concerns with aqueous buffers at higher temperatures is the fear of drying out the hybridization solution. As regards sensitivity, improvements are generally seen when you move from static to agitated hybridization. The use of a reciprocal shaker at a low speed setting ensures a wave-like motion and prevents localized target depletion. The ideal is an automated hybridization/fluidic station that ensures consistent hybridization and maximum sensitivity. — Gary Hardiman Genome Technology In our case, we are using the oligoarray system produced by Agilent Technologies. We just follow the recommended protocols from them. — Shoshi Kikuchi This is not a concern with Affymetrix microarrays as long as the sample quality is very good. — André Ponton That’s really a platform question. Affymetrix has made great strides in the last several years to clear up issues with their platform and get around the problems. For instance, there are only 25 base probes, but the 11 probes they now use on average per gene are very well selected. Some of the longer probe-based arrays do a better job, and NimbleGen and GE have some interesting products. For the longer 50-60mer probes, the signal to noise is better. But there are issues there too. All [platforms] inherently have a level of noise that I think most traditionally trained, reductionist biologists have to get comfortable with. There are a lot of variables, fairly involved methods, and there’s a degree of fluctuation that is inherent in the system. Some researchers have a lot of issues with that; they just don’t like that things are bouncing around a lot. The simplest solution I’ve had for that is I almost always go for the low-hanging fruit: I usually use a fourfold cutoff or greater and it cleans it up pretty quick. It’s one of the nice things about working on genomic-scale stuff — there are usually so many things to look for that it’s OK if you keep your stringency pretty high, because lots and lots of things will still meet that criterion. — Scott Tenenbaum Microarray Sample Prep Tech Guide 11 What tips do you have for clean-up and handling of arrays? A general precaution when dealing with fluorescent detection of arrays is to avoid getting any particles (which are usually highly autofluorescent) in contact with the arrays. Work in a dust-free environment; use only powder-free gloves; filter all solutions (0.22µm), and make sure they don’t get contaminated (discard any turbid solution); use double distilled water and eventually DEPCtreated. Centrifuge the probe before loading the upper part of it onto the arrays, especially if a column-based system was used upstream during the synthesis. Particles from the column are likely to be autofluorescent. — Philippe Demougin The investigator should avoid touching the arrays and avoid using powdered gloves, which often create fluorescent artifacts. A clean lab or dust-free area is essential. When washing arrays, the arrays should be fully submerged or edge effects will be seen. When drying arrays, I favor centrifugation over use of nitrogen. It’s also not a good idea to process too many arrays at once, as this can lead to noisy arrays. Arrays should be kept separate from each other. By this I mean not exactly side by side on a washing tray. It’s not a good idea to have fluorescent target being washed off one array and binding non-specifically to an adjacent array that is also being washed. — Gary Hardiman Normally [by] spraying the N2 gas. — Shoshi Kikuchi 12 In case of Affymetrix, recommendations. we follow their — André Ponton If you’re getting in on the field and it’s new to you, I would strongly encourage you [to] work with a core facility that has a lot of experience doing it. Core facilities that run one or two microarrays per month are really new at it themselves. You’d be better off working with people that run hundreds of arrays on a regular basis. They are much better at doing the technique and the analysis. I think people who spend a lot of time analyzing microarray data are much better suited to work with when you’re getting into this field. I have too often seen researchers who finally get enough money to run some arrays, but once they get their data back, they’re really on their own. They just don’t know what to do with the volumes of data one gets. There’s a little bit of hand holding, but not really enough. And so they don’t make any headway on what could have been some pretty good data. They spend a couple of months scratching their head and they eventually do exactly what I think they shouldn’t do, and that is: they take a few genes they’re interested in, and say, ‘OK, that gene I know about, so I’ll go focus on that.’ It defeats the whole purpose of doing this big genomic-scale analysis if you’re going to look at 30,000 genes and throw them all out to look at one. It seems kind of foolish. So, that would be my big suggestion: Get with a group that can give you the analysis support as well as run a lot of arrays, so they know what’s going on. — Scott Tenenbaum Microarray Sample Prep Tech Guide Genome Technology Q1: Which sample collection and storage techniques yield optimal RNA? (continued from p.7) avoid the noise of gene expression by cutting, etc., we normally prepare liquid nitrogen before tissue preparation. Then whole plant (control or stresstreated plants) will be frozen in liquid nitrogen and, after completely frozen, [the] RNA preparation process will be started. This kind of treatment can avoid unexpected gene expression during sample preparation process. As for the capture of specific tissue — such as the specific cell layer, specific organ — we will capture the tissue at normal temperature and, after capture, tissue will be frozen in liquid nitrogen. — Shoshi Kikuchi Usually it is best to keep the RNA in RNase-free water (commercial source) and put aliquots at -80°C. It is best to avoid multiple freeze-thaw cycles. — André Ponton Usually, we use Qiagen’s RNeasy prep or TRIzol extractions. I store my RNA in ethanol at -80°C. In my experience, you either have an RNase problem or you don’t. If you don’t, it’s pretty stable. And if you do, it doesn’t matter what you do — it’s gone. The trick is to not have any RNase problems. My RNAs are usually stable, especially at -80°C in ethanol, they’re good for a year, no problem. — Scott Tenenbaum Q3: How do you establish controls for heterogeneous or difficult samples? (continued from p.10) outlier becomes pretty obviously an outlier. Then you can either selectively remove it or lessen its impact. I think the biggest criticism of the field is most researchers will err on the side of doing more time points, rather than doing fewer time points and more replicates. So they’ll do one sample each at six different time points, as opposed to doing three replicates each at two time points. The data in the end is much more believable that way. — Scott Tenenbaum Genome Technology Microarray Sample Prep Tech Guide 13 List of resources Our microarray experts referred to a number of products, which we’ve compiled below. The recommended publications feature even more tips regarding sample prep and and experiment design. Products Affymetrix: GeneChip Arrays http://www.affymetrix.com/products/arrays/index.affx Agilent Technologies: Bioanalyzer http://www.chem.agilent.com/Scripts/PDS.asp?lPage =51 Oligo Array Kit http://www.chem.agilent.com/Scripts/PDS.asp?lPage =7307 Ambion: RNAlater http://www.ambion.com/techlib/resources/RNAlater/ index.html Bio-Rad Laboratories: Experion system http://www.bio-rad.com/ BD Clontech: Universal Total Reference RNA http://www.clontech.com/clontech/products/literature /pdf/brochures/UnivRNA.pdf GE Healthcare Life Sciences: Array products http://www.amershambiosciences.com/ NanoDrop: ND-1000 Spectrophotometer http://www.nanodrop.com/products.html NimbleGen Systems: Array products http://www.nimblegen.com/ 14 Qiagen: RNeasy Mini Spin Columns http://www1.qiagen.com/Products/RnaStabilization Purification/ TissueLyser http://www1.qiagen.com/Products/Accessories/Tissue Lyser/TissueLyser.aspx Stratagene: Reference RNA http://www.stratagene.com/products/showCategory. aspx?catId=193 Yasui Kikai: Multi-Beads Shocker (cell disruptor) http://www.yasuikikai.co.jp/company/e_index.html Publications Discovering Genomics, Proteomics, and Bioinformatics by A. Malcolm Campbell, Laurie J. Heyer (September 2002) Benjamin Cummings; ISBN: 0805347224 DNA Microarrays and Gene Expression by Pierre Baldi, Wesley G. Hatfield (October 2002) Cambridge University Press; ISBN: 0521800226 Microarrays and Cancer Research by Janet A. Warrington, Randy Todd, David Wong (June 2002) Eaton Pub Co; ISBN: 1881299511 Microarray Quality Control by Wei Zhang, Ilya Shmulevich, Jaako Astola (April 2004) John Wiley & Sons; ISBN: 0471453447 Applying Genomic and Proteomic Microarray Technology in Drug Discovery by Robert S. Matson (December 2003) CRC Press; ISBN: 0849314690 DNA Microarrays and Gene Expression by Pierre Baldi, Wesley G. Hatfield (October 2002) Cambridge University Press; ISBN: 0521800226 Microarray Sample Prep Tech Guide Genome Technology Advertisement High-Performance RNA for Microarray Analysis Reliable gene expression analysis using microarray technology depends strongly on the use of effective methods for preparing RNA from biological samples. Of critical importance is the preservation of the in vivo gene expression profile and the reproducible purification of intact RNA without any carryover of contaminants that can inhibit enzymatic reactions. We ask Stuart Pepper, a researcher with several years of experience in microarray analysis, to talk about obtaining high-performance RNA for reliable gene expression analysis. Stuart Pepper, Paterson Institute for Cancer Research, Manchester, United Kingdom We run a service for Cancer Research UK where users send us their RNA samples for microarray analysis. For users working with cell lines, we recommend purifying RNA using RNeasy® Kits and storing the purified RNA at –80ºC. RNeasy Kits fulfill the most important requirements for preparing RNA for microarray analysis, namely reliability and consistency of RNA yield. Over recent years, we have received more and more RNA samples purified from tissue. With tissue samples, collection and storage is an important issue. Also, tissue samples often provide more limited yields of RNA. To maximize RNA yields, stronger lysis conditions are needed. For this reason, we recommend our users to carry out RNA extraction using a high-quality phenol–guanidine reagent followed by RNA cleanup using RNeasy spin columns. In the past, RNA was always considered to be difficult to work with, but since we started to use RNeasy Lipid Tissue Mini Kit (box 1 of 2) RNeasy Kits, we have become more confident in handling RNA and have fewer problems with samples becoming degraded. In a recent experiment, we wanted to degrade some RNA for a test — we took RNA purified from an RNeasy spin column and left it on the bench for 24 hours. When we checked it on our Agilent® bioanalyzer, the RNA showed no sign of degradation at all! A broad range of QIAGEN® products cover all steps of the workflow for preparing highperformance RNA for microarray analysis, from sample collection and stabilization to sample disruption and homogenization and RNA purification. Products include RNAlater® TissueProtect Tubes, which offer convenient collection, stabilization, and storage of tissue samples without handling of liquid nitrogen. Also available are RNeasy Lipid Tissue Kits, which integrate optimal phenol-guanidine–based lysis of all types of tissue with purification of high-quality RNA using RNAlater TissueProtect Tubes RNeasy spin columns. Visit www.qiagen.com/geneXpression to find out more about standardized purification of high-performance RNA! Trademarks: QIAGEN®, RNeasy® (QIAGEN Group); Agilent® (Agilent Technologies, Inc.). QIAzol Lysis Reagent is a subject of US Patent No. 5,346,994 and foreign equivalents. “RNAlater®” is a trademark of AMBION, Inc., Austin, Texas and is covered by various U.S. and foreign patents. W W W. Q I A G E N . C O M Opt for innovation When Aff ymetrix needed unparalleled sensitivity and specificity for their next generation GeneChip® Exon Array, they opted for RiboMinus™ transcriptome enrichment from Invitrogen. At Invitrogen, we know that discovery thrives on innovation. That’s why our broad portfolio of innovative nucleic acid purification products offers the solutions you need: • TRIzol™ Reagent—the most popular and effective total RNA purification product on the market • ChargeSwitch® technology—high-throughput DNA and RNA purification without a centrifuge, vacuum, chaotropic salts, or solvents • PureLink™ nucleic acid purification kits—familiar spin column, 96- and 384-well plate, and gravity flow column formats from mini- to gigaprep scale • RiboMinus™ transcriptome enrichment—novel, negative selection system Affymetrix is thinking differently about nucleic acid purification—isn’t it time you did too? Opt for innovation. Opt for Invitrogen. Learn more at www.invitrogen.com/NAPrep. ©2005 Invitrogen Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (See the Invitrogen catalog or our website, www.invitrogen.com) Aff ymetrix, the Aff ymetrix logo, and GeneChip are registered trademarks, of Aff ymetrix, Inc. Products may be covered by one or more of the following patents and/or sold under license from Oxford Gene Technology: U.S. Patent Nos. 5,445,934; 5,700,637; 5,744,305; 5,945,334; 6,054,270; 6,140,044; 6,261,776; 6,291,183; 6,346,413; 6,399,365; 6,420,169; 6,551,817; 6,610,482; 6,733,977; and EP 619 321; 373 203 and other U.S. or foreign patents. For research use only. Not for use in diagnostic procedures.
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