Northern Exposure

Premade Northern Blots Researchers interested in gene expression have at their disposal a whole host of RNA profiling tools, including Northern blotting, microarray analysis, and RNase protection assays (RPA). Each of these techniques has advantages and disadvantages, and scientists must decide which approach will best suit their particular need. A Northern blotting experiment involves the electrophoretic resolution of RNA samples on a formaldehyde-agarose gel, transfer of the RNA onto a solid support (such as a nitrocellulose or nylon membrane), and the detection of specific transcripts using a labeled probe. RNA samples may be either total RNA (unpurified) or poly-A+ RNA (purified mRNA). Because mRNA represents only a small fraction of the total cellular RNA, Northern blots using total RNA samples require considerably more RNA than do those using poly-A+ RNA. One advantage of Northern blotting is the ease with which comparative analyses can be carried out, as researchers can run RNA samples representing different developmental time points, tissues, or organisms on a single gel. Therefore, a single blot can provide information about the developmental time course of gene expression in a particular organism or tissue, or it can allow for comparisons among tissues in one organism or between tissues of multiple organisms. Thus, Northern blot analysis can be used to address a variety of questions involving gene expression. This technique offers a number of advantages over both RPA and microarray analyses. It is relatively fast and easy, requires no special equipment or kits, and produces a wealth of both qualitative and quantitative data. Besides transcript abundance (which all three methods can detect), Northern blots can also detect the length of a specific transcript, the presence and relative abundance of splice variants, and the quality of RNA samples. Therefore, researchers studying gene expression patterns often find themselves contemplating at least one, if not many, Northern blotting experiments. Unfortunately, this powerful technique is also tedious and laborious and can cause researchers to waste valuable time at the bench with basic preparatory work. In addition, RNA work is notoriously difficult, as it is easy to contaminate samples with omnipresent RNases that will degrade the samples. The collection and processing of numerous RNA samples can therefore introduce additional parameters, such as RNA quality, into the experiment, creating possible problems with blot-to-blot variability. For these reasons, researchers often approach Northern blotting experiments reluctantly. Now, however, a number of companies have developed products to alleviate the difficulties associated with Northern blotting. Many of these companies offer ready-to-use total RNA or poly-A+ purified RNA samples, either stand-alone or transferred to positively charged membranes as premade Northern blots. The available tissue samples run the gamut from normal adult human liver to cancer cell lines to murine embryonic spleen. This article focuses on premade Northern blots and the applications to which researchers can put them. What's Normal? Courtesy of OriGeneOriGene HB2010 hybridized with beta-actin probe Often, researchers will want to determine where a particular gene is expressed in an adult animal. To make this determination the old-fashioned way, the scientist must harvest and process samples from each of the tissues to be tested, and then run and transfer the RNA gel. Now, companies such as DNA Technologies Inc. of Gaithersburg, Md., (ReadyNorthern™ RNA Blots) and Cirm Corp. of New Haven, Conn., (Cirm Northern Blots) offer a number of premade Northern blots, featuring either total RNA or mRNA samples from multiple tissues. DNA Technologies also sells Western blots that correspond with their Northern blots, so protein expression can be followed as well as RNA. OriGene of Rockville, Md., sells separate human and mouse 12-tissue Northern blots featuring poly-A+ RNA from brain, colon, heart, kidney, liver, muscle, placenta, small intestine, spleen, stomach, and testis. Ambion of Austin, Texas, gives researchers two different multi-tissue normal adult human blots (FirstChoice™), both of which contain brain, placenta, liver, spleen, and thymus poly-A+ RNA. Human blot 1 also contains skeletal muscle, heart, kidney, pancreas, and lung, whereas human blot 2 contains small intestine, colon, prostate, testis, and ovary. These blots can be purchased individually or bundled with Ambion's proprietary hybridization buffer (ULTRAhyb™), NorthernMax™ Low and High Stringency Wash Buffers, and Strip-EZ™ Probe Synthesis and Removal Kit, which allows these blots to be reused up to 15 times. Several companies offer premade blots designed for researchers interested in examining the specifics of a particular organ system. For example, Stratagene of La Jolla, Calif., offers a MessageMap™ Northern Blot specifically devoted to the female reproductive system, with RNA from cervix, ovary, placenta, uterus, and vulva. Palo Alto, Calif.-based BD Biosciences-CLONTECH's collection of Multiple Tissue Northern (MTN ® ) Blots includes premium poly-A+ RNA human samples from tissues of the brain and muscle, along with the endocrine, cardiovascular, immune, and digestive systems. The Human Brain II, III, and IV Blots cover the cerebellum, cerebral cortex, medulla, spinal cord, occipital lobe, frontal lobe, temporal lobe, putamen, amygdala, caudate nucleus, hippocampus, total brain, substantia nigra, corpus callosum, subthalamic nucleus, and thalamus, thus offering scientists a large, representative view of the human adult central nervous system. All Grown Up Courtesy of ViroGenViroGen Northern Blot A major concern of modern biological research focuses on genes implicated in development, both because of their role in organ and tissue formation, and because of the important roles these genes play in the generation of birth defects. In order to determine the role a particular gene might play in the development of a specific tissue or organ, a researcher must precisely excise that tissue from multiple embryos of varying ages. This is a difficult, time-consuming, and expensive task, especially if mammalian samples are being used. The acquisition of human embryonic tissue in particular often introduces additional complications. 1,2 To alleviate some of these problems, ViroGen of Watertown, Mass., offers human development RNA blots by stage (six stages, weeks six through twelve) and tissue (heart, kidney, muscle, brain, liver, lung, and whole embryo). Similarly, BD Biosciences-CLONTECH and Cirm carry both a human fetal blot, which consists of fetal brain, lung, liver, and kidney, and a murine embryonic blot with tissue from 7-, 11-, 15-, and 17-day embryos. Finally, Geno Technology of St. Louis offers human embryonic messageHUNTER™ Northern Blots with mRNA from brain, heart, kidney, liver, lung, and whole embryo from different stages of development between six and twelve weeks. The ResGen Northern Territory™ line of human RNA blots from Carlsbad, Calif.-based Invitrogen Corp. offers a large selection of both mRNA and total RNA blots, including two fetal RNA products. The mRNA blots in this line are prepared using either of two different mRNA normalization methods. The Act-N™ mRNA Blots are normalized to equal b-actin levels, enabling the data to be compared to previously published blots that were also normalized to b-actin. On the other hand, the REAL™ mRNA Blots (mRNA, Equal Amount Loaded) are prepared with equivalent mass quantities of RNA (2 mg) loaded per lane. The two fetal human blots currently included in the Northern Territory line are the Fetal Human Brain mRNA blot, containing samples from the frontal, temporal, parietal, and occipital lobes, plus an internal adult lung sample enabling blot-to-blot comparisons, and the Fetal Human total RNA blot, which contains RNA from the thymus, adrenal gland, colon, and skin. There are a number of products designed to meet the needs of scientists interested in large-scale surveys of gene expression. For example, ViroGen carries a 42-lane blot (seven tissues by six stages) to cover many aspects of human development. Invitrogen's ResGen Northern Territory human RNA blots feature total RNA and mRNA from 40 normal and cancerous adult tissues (including multiple tissues from the central nervous and gastrointestinal systems) and eight fetal tissues preblotted on a positively charged nylon membrane. For scientists needing more than 40 samples, there is the Human Multiple Tissue (MTE™) Array from BD Biosciences-CLONTECH, with mRNA from 60 adult human tissues, eight cancer cell lines, and seven fetal human tissues, as well as eight internal controls. When Something Goes Wrong As humans are naturally concerned with their own mortality, much of the effort of biological research is focused on understanding, and thereby repairing, the mechanisms of human disease. Of particular interest, therefore, is the identification and characterization of the genes responsible for diseases such as Alzheimer's and different types of cancer. The majority of premade Northern blots available in this category focus on the study of cancer, due in large part to the multitude of cancerous cell lines available. Preliminary survey-type studies can be aided by blots derived from RNA of basic human cancer cell lines, which a number of companies offer. For example, Ambion's Human Blot III features mRNA from acute T-cell leukemia (Jurkat), Burkitt's lymphoma (CA46, Namalwa, and Raji) breast carcinoma (MDA-MB-453), epidermal carcinoma (A-431), uterine carcinoma (MES-SA), osteosarcoma (MG-63), diploid lung (MRC-5), and cervical cancer (HeLa S3) lines. Stratagene's MessageMap Cancer Cell Line Blot No.1 also features RNA from the human cell lines HeLa S3, Raji, and MDA-MB-453, as well as HepG2 (heptoma), HL-60 (promyelocytic leukemia), Ntera (teratocarcinoma), PC-3 (prostate adenocarcinoma), and U937 (histiocytic lymphoma). Invitrogen carries a number of options to analyze gene expression in cancerous tissues. The ResGen REAL human tumor panels (four blots available) are prepared from mRNA isolated from two different normal and cancerous tissues, whereas the REAL human single-tissue, multi-sample tumor blots enable comparisons of tumor mRNA from three different donors (nine blots available). The company also offers a large selection of total RNA human tumor blots (seven blots available) with RNA from four different donor tissues on each blot, to facilitate the study of the expression of general tumor genes. The majority of these panels include tissue samples from groups of organs commonly affected simultaneously; Northern Territory Human Tumor Panel Total RNA Blot IV, for example, contains total RNA from breast, uterus, fallopian tube, and ovary. Not all disease-related Northern blots deal with the study of cancer. Genpak Inc. of Stony Brook, N.Y., (Touch Blot™ Northern Blots) carries two blots featuring RNA from different brain regions from Alzheimer's patients, in addition to a large collection of normal tissue and tumor blots. These blots consist of total RNA isolated from seven functionally distinct regions of human brains from patients afflicted with Alzheimer's disease, including the frontal, temporal, and occipital lobes, amygdala, and hippocampus. For examination of these tissues on the cellular level, Genpak also offers slides prepared from the same tissues represented on the blots for in situ hybridization or immunohistochemistry, thus offering researchers interested in the genes regulating Alzheimer's disease a complete package. In Reverse The majority of products discussed above are designed to ease the burden of RNA analysis by freeing the researcher from the hassle of collecting various tissues, harvesting RNA, and preparing their own blots. Because only a single gene can be assayed at one time in a Northern assay, these products are really intended for the researcher focusing on the expression patterns of a small number of genes in a large number of tissue types, or throughout development. However, until recently, scientists interested in examining the expression patterns of multiple gene families in just one or two tissues had to do things the old-fashioned (and therefore painstakingly slow) way. But now, U.S. Biological of Swampscott, Mass., has developed an innovative line of products to aid these frustrated researchers. Instead of membranes consisting of RNA samples from multiple tissue types, the company's GeneScreen ® mRNA Blots contain RNA from multiple gene members of a particular family that can then be probed for expression in a specific tissue. This "reverse" Northern analysis is akin to microarray analysis, and it offers scientists the chance to examine all of the known gene members in families such as the JAK/STAT pathway or the MAP kinase pathway simultaneously in whichever tissue is the focus of their research. To use these blots, the researcher prepares a probe by incorporating a label during synthesis of cDNA from the total RNA prepared from the tissue of interest. 3 The company offers more than 30 gene family options ranging from genes involved in apoptosis to endocrinology-related genes. A Custom Fit Despite the abundance of premade Northern blots on the market, some researchers still find that their specific blotting needs are not being met. These scientists, however, do not have to worry that their only option is to start collecting RNA samples and pouring formaldehyde gels; a number of companies now offer custom services to provide researchers with any blotting combination they might need. Both Molecular Diagnostic Services Inc. of San Diego and Lofstrand Labs Limited Inc. of Gaithersburg, Md., offer complete Northern hybridization analysis services. Researchers need only specify the tissues and developmental stages of interest and then wait for the results, which include hybridization, filter autoradiography, and densitometry analysis. These companies also allow scientists to choose individual options. For example, researchers can just have RNA isolated for them or can opt to receive RNA transferred to a nylon membrane (up to 20 lanes offered by Lofstrand), instead of choosing the full-service Northern blot option. Commonwealth Biotechnologies Inc. of Richmond, Va., offers custom services to meet the needs of scientists for whom Northern hybridization analysis represents just one part of a larger project. This company not only provides Northern blot services ranging from RNA isolation through the reprobing of membranes, but also works with researchers to provide any other experiments needed to complete the project. In this age when molecular mechanisms are being deciphered at an astonishing pace, determining gene expression patterns has never been easier. Gone are the days of tedious tissue collection and RNA processing, when problems with RNA integrity appear only after multiple-day experiments. Instead, premade Northern blots ensure that high-quality gene expression data can be easily and rapidly obtained, even by labs not specializing in RNA work. Deborah Stull (zebrafish9@yahoo.com) is a freelance writer in Lafayette Hill, Pa. References 1.T. Agres, "On the brink," The Scientist, 15[11]:1, May 28, 2001. 2. E. Russo, "Cultivating policy from cell types," The Scientist, 15[11]:6, May 28, 2001. 3. D. Stull, "A feast of fluorescence," The Scientist, 15[10]:20, May 14, 2001. Suppliers of Premade Northern Blots Ambion (800) 888-8804 www.ambion.com BD Biosciences-CLONTECH (800) 662-2566 www.clontech.com Cirm Corp. (734) 459-0082 www.cirmcorp.com Commonwealth Biotechnologies Inc. (800) 735-9224 www.cbi-biotech.com DNA Technologies Inc. (800) 998-3628 www.dnati.com Genpak Inc. (800) 385-9248 www.genpakdna.com Geno Technology (800) 628-7730 www.genotech.com Invitrogen Corp. (800) 533-4363 www.invitrogen.com Lofstrand Limited Inc. (800) 541-0362 www.lofstrand.com Molecular Diagnostics Services Inc. (858) 259-0301 www.mds-usa.com Origene (888) 267-4436 www.origene.com Stratagene (800) 424-5444 www.stratagene.com U.S. Biological (800) 520-3011 www.usbio.net Virogen Corp. (888) 847-6436 www.virogen.com

Northern Exposure

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