            gerona      J Gerontol A Biol Sci Med Scigerona      The Journals of Gerontology Series A: Biological Sciences and Medical Sciences      J Gerontol A Biol Sci Med Sci      1079-5006      1758-535X              Oxford University Press                    41310.1093/gerona/60.4.413                        Journal of Gerontology: Biological Sciences                            National Institute on Aging Microarray Facility—Resources for Gerontology Research                                          Nadon            Nancy L.                                1                                                          Mohr            David                                2                                                Becker            Kevin G.                                3                          1Office of Biological Resources and Resource Development, National Institute on Aging, Bethesda, Maryland.        2The Johns Hopkins University Microarray Facility, Baltimore, Maryland.        3Gene Expression and Genomics Unit, National Institute on Aging Gerontology Research Center, Baltimore, Maryland.                    Address correspondence to Nancy L. Nadon, PhD, Office of Biological Resources and Resource Development, National Institute on Aging, 7201 Wisconsin Ave., GW2C231, Bethesda MD 20892. E-mail: nadonn@nia.nih.gov                    4        2005            60      4      413      415                        23          11          2004                          28          9          2004                            Copyright 2005 by The Gerontological Society of America        2005                    The use of DNA microarrays allows rapid, large-scale analyses of changes in gene expression. This article introduces the National Institute on Aging Microarray Facility, developed to provide DNA microarrays to the biogerontology research community and to promote the use of this new technology.                              hwp-legacy-fpage          413                          hwp-legacy-dochead          RAPID COMMUNICATION                                      MICROARRAYS, hybridization platforms containing large numbers of complementary DNA (cDNA) clones or gene-specific oligonucleotides, have revolutionized gene expression analysis. They allow analysis of expression of from hundreds to thousands of genes in a single hybridization experiment. These high-throughput analyses have contributed enormously to discoveries that might never have been made in the single-gene analysis approach. Arrays have increased the pace of discovery in the following areas of cancer research: cancer development, normal aging, and pathological development (1,2).      The DNA Array Unit of the National Institute on Aging Gerontology Research Center (NIA GRC) has focused on the development of microarrays on nylon membranes. Membrane-based hybridization technology has a well-established place in modern molecular biology laboratories. Any laboratory doing standard hybridization protocols, such as Northern and Southern blotting, has the basic expertise and equipment required to use membrane microarrays. Radioactive labeling reagents are highly sensitive, widely available, and relatively inexpensive. Membrane arrays have some advantages over chips or glass arrays, particularly for laboratories that do not want to make a significant investment in new technology and equipment. Both probes and membranes can be reused, reducing the cost of replicating experiments (3). Although membrane arrays do not lend themselves to two-color probing that can be done on glass arrays, analytical software for membrane arrays is as powerful as that used with glass arrays. Large numbers of membranes can be hybridized at a time, increasing throughput, sample sizes, and thus overall experimental design. Membrane arrays, and the reagents to use them, are quite often less costly than commercially available array formats.      To make membrane arrays more accessible to the research community, the NIA Array Unit established the NIA Microarray Facility to supply nylon membrane cDNAs at low cost. The website for the NIA Microarray Facility, located at Johns Hopkins University, is: http://www.daf.jhmi.edu/microarray/index.htm.              Mouse cDNA Microarray      The mouse 17K cDNA microarray contains 16,897 features, which include approximately 12,341 unique genes. The mouse array was based on the original 15K mouse cDNA clone set developed from mouse embryos at the NIA (4,5). Approximately 1700 genes were added to the original set to produce the 17K sequenced, verified clone set for production of this array format. The NIA mouse 17K array represents a large general mouse array similar to other large cDNA array collections. Although it is not a whole genome array, it contains a set of clones from embryonic cells and tissues from approximately 34 unique cDNA libraries, including specific libraries from neonatal tissues. Gene identification numbers in the 15K mouse clone set are available from the NIA Microarray Facility website (from the downloads link), and are also posted at the NIA Laboratory of Genetics website (http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html). The mouse cDNA microarrays are now available.      Figure 1 shows a representative set of NIA mouse 17K membranes. Each microarray consists of two nylon membranes, each approximately the size of a 96-well culture plate, or 8 cm × 12 cm. Earlier versions of these arrays have been used productively in many model systems, including murine models of drug abuse (6,7) and murine developmental models (8,9).              Human cDNA Microarrays      Human cDNA microarrays have been developed and are currently available. This human array consists of full-length cDNA clones generated by the National Cancer Institute's Mammalian Gene Collection (10) (http://mgc.nci.nih.gov/). These clones have been completely sequenced and have been specifically chosen to represent the full-length open reading frame of each representative gene. The human cDNA arrays developed from these clones consist of 9600 features, representing approximately 6424 unique genes. This is printed on one 8 cm × 12 cm filter. Figure 2 shows a representative example of the human NIA membrane.              Gene Expression Analysis Using the NIA Microarrays      The range of biological applications, laboratory use, data collection, and analysis strategies using this array format is conceptually similar to most other array formats. Array hybridization is straightforward and requires minimal training time. Radioactive labeling for these arrays with 33P-α-dCTP requires a relatively low amount of input RNA (5 μg) of total RNA per sample, with no nucleic acid amplification, (i.e., RNA amplification). This quantity is particularly amenable to small tissue samples. Using this approach, we have utilized membrane arrays in this format quite extensively in studies of mouse embryogenesis, in in vitro cell line studies, and in studies of drug toxicity, normal aging, caloric restriction, and Alzheimer's disease, among many others. These arrays produce single channel data, similar to Affymetrix Genechip arrays or those using Illumina BeadChips. This produces primary intensity values, which are normalized and ultimately compared against other experimental samples to produce ratio data. The data are then analyzed using approaches and bioinformatic tools very similar to most other microarray formats. There are no specific plans for a central repository for data from these arrays. It is recommended that data should be deposited in a public microarray repository such as the National Center for Biotechnology Information's Gene Expression Omnibus (NCBI GEO). The data-tagging process in GEO should allow identification and retrieval of all information derived from this series of arrays.              Information and Support Resources      The NIA Microarray Facility website contains several links to information useful in the design and execution of experiments using the membrane arrays. The downloads link takes the viewer to information on the gene identification numbers of the clone sets used on the arrays. The images link presents views of hybridized arrays. The FAQ (frequently asked questions) link provides answers to questions often encountered by users new to nylon array hybridization and additional information on the versatility of the nylon arrays. The references link lists references that demonstrate the range of work possible with nylon arrays.      There are protocols for preparing probes for hybridization, probe labeling, and reusing the membranes on the protocols link. All protocols listed were developed, tested, and used extensively by the NIA Array Unit. One of the benefits of radiolabeled hybridization of nylon membranes is that several options for data management and analysis already exist.      The analysis link provides information on analysis of the array data, including information on analytical software and how to use it. The analysis link includes links to many free analysis programs that can be downloaded from the Web.      Assistance with the use of NIA arrays or purchasing arrays is available from the contacts link and may also be sought by e-mailing the authors. Pertinent addresses areKevin Becker, PhD: beckerk@grc.nia.nih.govNancy Nadon, PhD: nadonn@nia.nih.govNIA Microarray Facility home page: http://www.daf.jhmi.edu/microarray/index.htm.                                      Decision Editor: James R. Smith, PhD                          Figure 1.                      Typical images from the National Institute on Aging Mouse 17KA and 17KB filters                                              Figure 2.                      Typical image from the Human MGC 9.6K filter                                                    References              1        Fan J, Yang X, Wang W, Wood WH, Becker KG, Gorospe M. Global analysis of stress-regulated mRNA turnover by using cDNA arrays. Proc Natl Acad Sci U S A.2002;99:10611-10616.                    2        Sawiris GP, Sherman-Baust CA, Becker KG, Cheadle C, Teichberg D, Morin PJ. Development of a highly specialized cDNA array for the study and diagnosis of epithelial ovarian cancer. Cancer Res.2002;62:2923-2928.                    3        Donovan DM, Becker KG. Double round hybridization of membrane based cDNA arrays: improved background reduction and data replication. J Neurosci Methods.2002;118:59-62.                    4        Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, et al. Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. Proc Natl Acad Sci U S A.2000;97:9127-9132.                    5        Kargul GJ, Dudekula DB, Qian Y, Lim MK, Jaradat SA, Tanaka TS, et al. Verification and initial annotation of the NIA mouse 15K cDNA clone set. Nat Genet.2001;28:17-18.                    6        Xie T, Tong L, McCann UD, Yuan J, Becker KG, Mechan AO, et al. Identification and characterization of metallothionein-1 and -2 gene expression in the context of (+/−)3,4-methylenedioxymethamphetamine-induced toxicity to brain dopaminergic neurons. J Neurosci.2004;24:7043-50.                    7        Noailles PA, Becker KG. Wood WH 3rd, Teichberg D, Cadet JL. Methamphetamine-induced gene expression profiles in the striatum of male rat pups exposed to the drug in utero. Brain Res Dev Brain Res.2003;147:153-162.                    8        Buttitta L, Tanaka TS, Chen AE, Ko MS, Fan CM. Microarray analysis of somitogenesis reveals novel targets of different WNT signaling pathways in the somitic mesoderm. Dev Biol.2003;258:91-104.                    9        Suemizu H, Aiba K, Yoshikawa T, Sharov AA, Shimozawa N, Tamaoki N, et al. Expression profiling of placentomegaly associated with nuclear transplantation of mouse ES cells. Dev Biol.2003;253:36-53.                    10        Strausberg RL, Feingold EA, Grouse LH, Derge JG, Klausner RD, Collins FS, et al. Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc Natl Acad Sci U S A.2002;99:16899-16903.            