The Biotechnology Education Company ® DNA/RNA Microarrays Storage: Store the entire experiment in the refrigerator upon receipt. EXPERIMENT OBJECTIVE: ‘The objective ofthis experiment is to understand the ‘basis of microarrays as applied to functional genomics. This simulation s designed to provide students with the ‘opportunity to analyze differences in gene expression using a two-color microarray. EDVOTEK, Inc. » 1-800-EDVOTEK + www.edvotek.com| EDVO-Kit #235 DNA/RNA Microarrays fr? EDVOTEK. | Table of Contents } | rage Experiment Components 3 | experiment Requirements 3 Background information 4 | experiment Procedures Student Experimental Procedures 3 Experimental Results and Anais 10 study Questions 10 J tnstructors Gude | peetab Preparation " i Expected Results 1B | study Questions and Answers 14 Visit Us Online! otek.com Download dvotek kts with Protocols! Browse Order ouronline |} Products catalog! Receive Technical Support EDVOTEK and The Bitechnoloy Edvaton Company sre registered trademarts of EOVOTEK, In \ The Blotechnology Education Company ® + 1-800-EDVOTEK + www.edvotek.comrece a 3 it #235 DNA/RNA Microarrays) } Experiment Components r | ‘+ Four different Microarray sample QuickStrips™ This experiment = Patient one is designed for “Patient two 10 groups “Patient three = Patient four store entre + Microarray Card experiment in the refrigerator upon receipt. Requirements ‘+ Fixed:-volume (5 i) or variable volume micropipets + Pipet tips Long wave UV. light source research ony. They ae not 1 be used fr diagnostic oF dug purposes nar ad ministered oor consumed Alomponens #e Intend edna | Geeta an a, 4 d EDVOTEK - The Biotechnology Education Company® EDYOTEKe 1.800.EDVOTEK + www.edvotek.com \ 24-hour FAX: 202.370.1501 + email: Info@edvotek.com =m4 — EDVO-Kit #235 DNA/RNA Background Information UNLOCKING THE HUMAN GENOME ‘An organism's genome contains the genetic information necessary for its growth, development, and survival In humans, this information is contained ‘within 23 pairs of chromosomes contained within a cell's nucleus. In the ‘early 1990s, researchers resolved to sequence the entire human genome (six billion base pairs of DNA). Tis international undertaking, called the Hu- man Genome Project, launched the field of “genomics” (the study of the sequence and structure of the genome). As a result of the Human Genome Project, avast amount of information about the DNA sequence has been made publicly available. ‘After the complete consensus sequence of the human genome was pub- lished in April 2003, scientists began investigating the information hidden ‘within the DNA sequences. Using the sequence information, specific genes ‘an be mapped to their chromosomal location, and novel genes are still be- ing identified today. However, sequence analysis has determined that there are only 21,000 protein-producing genes in the human genome, a number ‘much lower than estimates made before the Human Genome Project. Background Information Data from the Human Genome Project has shown that DNA sequences only differ approximately 0.2% between individuals roughly one base in every 500 is changed). Specific variations in an individual's genome can be used as markers to predict predisposition for particular diseases. Scientists can ‘analyze these genetic differences to explore human diversity and evolution ‘at the DNA sequence level. In addition to DNA sequences that cade for pro- teins, the genome includes DNA sequences that influence protein production via other mechanisms. For example, sequences known as promoters control ‘canscription ofa specific mRNA. Other ONA sequences code for ribosomal RNA, transfer RNA, and microRNA, which work together to regulate trans- lation of proteins. For these reasons, a fundamental understanding of the entice human genome sequence is critical, even if a majority of the genome does not code for proteins. ANALYZING GENE EXPRESSION USING MICROARRAYS Precise regulation of gene expression is essential for the normal function (of cells and tissues. Depending on the characteristics of its promoter, the ‘expression of a particular mRNA may vary from no expression to hundreds Cf copies per cell Traditional techniques such as Northern blotting analyze ‘the expression of only a few genes at a time, making genomics research very ‘time consuming. The emergence of DNA Microarray technology has made it possibie to produce and analyze data measuring the levels of mRNA from ‘thousands of genes in a single experiment. ‘Microarrays (or “gene chips") have made it possible to identity, classify, and assign functions to many uncharacterized genes, simply by determining when the genes are expressed or repressed. Their small size and ability to ——————— EDyOtEK. Soph soso SEOVOTER hes aight SSS Sa om “cosons The Biotechnology Education Company ® + 1-800-EDVOTEK + www.edvotek. com smnthe Background Information Figure 1: ‘An example of Gene Chip. Duplaton of any part of his document permitted fer nan protit educational purposes ony Copyright © 20082019 EOVOTEK neal igh served Tso? analyze expression from large number of genes simultaneously have made microarrays an important tool for genomics research in diverse fields such as ‘drug discovery, toxicology, and medical diagnostics, Each chip consists of short, single-stranded pieces of DNA called oligonucle: tides (or oligos) affixed to a glass slide (Figure 1). The chip contains a grid comprising thousands of oligos, each with a known sequence that corre- sponds to a particular gene to be analyzed. Because a single chip contains thousands of spots, each experiment can accurately analyze the expression levels of thousands of genes. ‘Two-color microarray technology allows a comp: profiles between two different samples (for example, normal skin cells versus, skin cancer cells). There are four basic steps involved in a two-color microar- ray (Figure 2) The color and intensity of fluorescence at each particular spot allows researchers to identify key genetic dferences, such as those between ‘normal skin ces and skin cancer cells. The control Oo Oo "0 "0 "O 70 °O =O ries EL forall four patients: OCOOO00000 en, © PEF SH) spats €- Hon the micro- SSoo00g0] maven 4, PLACE the membrane in a 37°C incubator for five minutes to allow the samples to dry. 5, VISUALIZE the microarray using a long-wave handheld UV transilumina- tor, 6. (Optional) RECORD your results by photographing the microarray card Using either a Digital or a Polaroid camera = Recommended camera settings are aperture f 5.6 for 2 seconds. ‘the photograph is too light, change the aperture to f 8 and ex- pose for 2 seconds. + Hfto0 dark, keep the aperture at 5.6 and reduce the shutter speed to 1 second, Specific settings will vary depending upon the photodacumentation system you are using. For additional information, refer to the instructions included ‘with your photodocumentation system, Copyright ©2008 3013 EDVOTEK neal ight served "Bs 302? Nuss The Biotechnology Education Company ® + 1-B00-EDVOTEK + www.edvotek.com roa optus unnonunapgon ~ byoir. on x o @ 2. 3 2 3 = ¥ 3 a ® 2 5 8 aEDVO-Kit #235 DNA/RNA Microarrays 10 Experimental Results and Analysis Using the appropriate symbols, record your results in the sample micro- array card (see key below right) i °O =O °O °O "O 70 °O =O KE} @ = normal (yellow) © = up regulated (red) @ = down regulated (green) © = no expression (black) z> >O 70 °O °O "O "Oo 20 =O Oo O °0 20 "O 70 20 =O w g 5 Ss 3 g 2 = ss = o E 3 a x iS B> je >O 20 °O °O "O 70 20 =O Study Questions ‘Answer the following study questions in your laboratory notebook, 1. What new information has become available as a consequence of the human genome project? 2. Explain the core technology behind microarrays and whiy is it impor- tant for biotechnology and medicine. 3. How are cDNAS libraries made? A. What information has DNA microarrays made possible? 5. How are the individual spots on a microarray chip identified and analyzed? Duplation of any part ofthis document permitted for non-profit educational prpotsony, Copyright © 2008 S0r3 EDVOTEK nal gh rseve "sr lotechnoloay Education Company ® + 1-800-EDVOTEK + www.edvolek.com