1 GSR Microarrays Project Management System A User s Guide GSR Microarrays Vanderbilt University MRBIII, Room st Avenue South Nashville, TN (615)
2 Introduction Microarray experiments can be a daunting prospect. From the very beginning, investigators must make informed decisions about a technology they may be unfamiliar with ( Which platform will be best for me? How many arrays should I hybridize? ). After all these questions have been answered and the experiment completed, the task gets even more intimidating. It is not uncommon for an experiment to produce over a million points of data, and a strong understanding of statistics, as well as biology, is necessary to extract meaningful results. At GSR Microarrays, our goal is to make this technology accessible to all investigators. At every step of the process, we provide expertise and information to allow investigators to make the best decisions for their experiment. In order to maintain the high quality of work investigators have come to expect from us, as well as effectively communicate with an ever-growing list of clients, GSR Microarrays has developed a web-based project management system. In addition to streamlining the order submission process, this website provides a central source for all data relating to a project, an electronic lab notebook so that investigators know every step from submission to analysis, and allows investigators to add new collaborators to their project and interact with them. GSR Microarrays Projects GSR Microarrays uses projects to organize sample submission and data analysis. All hybridizations performed within a project must be directly comparable to each other. Therefore, if your experiment requires any non-comparable changes, such as a change in microarray technology, study tissue or organism, you must create multiple projects. Multiple projects may exist for a single investigation if any of the following occur: Change in fundamental hypothesis Change in array technology (i.e., GSR Microarrays cdna => Affymetrix) Change in RNA amplification requirements (amplified & non-amplified samples should not be compared in the same project) Change in study organism or study tissue GSR Microarrays currently offers six general classes of services: 1. Gene Expression Microarrays (including microrna arrays) 2. DNA Genotyping Microarrays (including Mapping, and Resequencing arrays) 3. Real-time PCR 4. RNA Quality Control 5. RNA Amplification 6. RNAi/cDNA clone distribution The first three service types require careful experimental design and a number of detailed decisions that an investigator who is inexperienced with the technology may feel uncomfortable making. Therefore, we recommend that users consult with a staff member by setting up an appointment prior to creating a project for one of these services. This will give the user the opportunity to review the platforms offered at GSR Microarrays and choose the best platform for their needs. It will also help the user avoid some common
3 mistakes in designing experiments, as well as allow GSR Microarrays to anticipate new submissions and minimize turnaround time. The latter three service types (RNA Quality Control, RNA Amplification and RNAi/cDNA distribution) are simply informational in nature, and do not require an experimental design. Therefore, we do not require prior consultation to create a project for these services, and can be created by clicking the appropriate link on the Create New Project page. A project is not required to request RNAi/cDNA clones. Creating an Account 1. Go to GSR Microarrays Web site at 2. Click on the Create New Account link on the right side of the page. 3. Fill out the requested registration information. Make sure that the address you supply is correct, and that you choose a username. 4. Click the Register button. Account Activation 1. Check your . In a few minutes, you should receive an from with the subject GSR Microarrays Registration Activation. 2. Click on the link in the Choose a password for your account. Enter it twice once in each field of the web page (this will help prevent typos). 4. Click the Activate Account button. You should see a web page informing you that your account has been activated.
4 Project Pages Once you have registered and selected a password, you can log in by entering your username and password on the right side of any GSR Microarrays web page. After you log in, the right side bar will change to a new menu that you can use to access project-specific information. My Projects This is the first page you will see when you log in. From here, you can get a quick overview of all of your projects. My Settings This is where you can change any of the information that you supplied at registration if your phone number or address changes, this page is how you can let us know. You can also change your password or (un)subscribe to mailing lists here. Create New Project Users who have begun investigating a new hypothesis can create a new project here. See Creating a New Project (below) for more information. Feedback If there s anything you like or don t like about these pages, or if you encounter any errors, visit the Feedback page. You will be given a direct line to your comments to the website developer.
5 Bioinformatics Tools This page lists some of the tools we ve developed that our clients may find useful. Currently, there are four. From this page, you can: Find homologs between organisms based on Locus Link ID Renumber annotations from 384-well to 96-well format (and vice versa) Retrieve a batch of probesets on the Affymetrix Human chips Search for publicly annotated genes on any of our most popular microarrays. Logout When you re finished using the GSR Microarrays website especially on a public computer click here to log out of your account. Creating a New Project The first step in creating a new project is to specify the Primary Investigator. The PI is the person who is financially responsible for your experiments. They will be notified by of all work requested of GSR Microarrays. Because we need to be able to contact them, the PI must have a GSR Microarrays account before the project can be created. After clicking on the Create New Project link, you will be asked to specify the PI for your new project. Enter the PI s name into the text boxes and click the Search button. Find your PI in the list that appears, and click the Set link next to their name. The next page will ask you to specify the details of your project. These include: Title: A short, descriptive sentence to help you distinguish this project from any others you may create. PI: This displays the name of the person you chose on the previous screen. If you made a mistake there, you can always click on the Change link here. The following fields are displayed if you are creating a microarray, Real Time PCR, or RNA Amplification project:
6 Project Type: The general technology type used for this project, such as Gene Expression Microarrays, Genotyping, acgh, Epigenetics, Real-Time PCR, High Throughput Sequencing, or RNA Amplification. Array Type: Only shown if you have a microarray project. The specific Manufacturer platform (or, for a Genotyping project, size) of the chip you plan to use. Abstract: This will help us to understand your experimental design. Different tissue types and experimental treatments can radically affect the performance and results of a microarray experiment and knowing about them ahead of time can help us perform your experiments and analysis with much better results. Please provide as much detail as you would for a poster abstract, and be sure to spell out acronyms. Users who create projects without a detailed abstract will not be allowed to submit samples. Once you ve filled in all of your information, click the Create New Project button. Working with Your Project Once you have a project (either by creating a new one or being added to an existing one), you can access the project page at any time by clicking on the project number. From the project page, you can view all of the data associated with your project and submit either samples or hybridizations. The section below will discuss the project page for a microarray project. The other project types have fundamentally similar project pages, and differences will be pointed out in the text. Project Header The top section of the project page displays summary information for the project number, title, abstract,
7 PI, etc. The only details in this section that can be changed are the Abstract and the list of Contacts. Any member of the project may edit these fields by clicking on the appropriate link beside them. Be very careful when making edits, however it is possible to remove yourself as a Contact, and then find yourself unable to correct your mistake. Samples This section only exists in Quality Control and Microarray projects and contains every sample you ve submitted to VMSR since April (To learn how to submit new samples to GSR Microarrays, see the Submitting Samples section of this manual.) The samples in the Pending section have been submitted by you, but have not yet been assessed by GSR Microarrays, so only the information that was supplied at the time of submission is available to view. The Ready to Use section contains all samples that have been assessed for quality by GSR Microarrays and are now available for hybridization. A summary of our QC results is available here, although investigators should read the detailed results in the Results section (see below) before making a final judgment about using their sample. Samples will move to the Completed section after requested hybridizations have been scanned and data have been posted. Hybridizations This section only exists in Microarray projects, and lists the hybridizations that have been requested. Once a particular hyb moves from the In Progress list to the Completed list, the results for that hybridization can be found in the Results section. Comments This section is your electronic lab notebook. Whenever a GSR Microarrays staff member does any work on your project, they will make an entry in this section. Project members may also post their own comments, which is mostly useful if project teams are geographically spread out. If you have a question or concern about your project, be sure to contact a GSR Microarrays staff member directly as user comments are not monitored. Investigators are encouraged to check for new GSR comments regularly, as it is the best way to stay up-to-date on your project s progress. Results This section contains all of the files and data associated with your project. The files are categorized: Quality Assay- Any data generated while assessing samples for quality and quantity. Includes Bioanalyzer reports, Pico Green assay results, Gel Images, and NanoDrop logs. Experimental Records- Files that describe what procedures were done. A copy of your order form is placed here, as well as detailed worksheets for labeling and hybridization. Raw Data- Unanalyzed, untransformed data generated directly by the scanner. Any analysis must start with this data. Preliminary Analysis- A first-pass, rough analysis performed by GSR Microarrays. This is usually the direct, two-array comparison that is offered with single-color hybridizations. All data in this category is preliminary, and must be verified through other methods. GSR Analysis- Any advanced data analysis performed by the GSR. The methods and parameters used to create these files will be listed in the description of the file, or in the Comments section.
8 Other- Any files that did not fit in one of the other five categories. Submitting Samples Note: DNA Microarray Projects do not require a separate step for requesting hybridizations. Submitted DNA is assessed for quality and quantity; however, all submitted DNA samples are hybridized to the chip specified at project creation, unless the PI specifies not to hybridize the samples, based on QC/QA of the samples. This section describes the sample submission procedure for Quality Control, and Microarray projects. Realtime PCR projects do not yet use the electronic database for sample submission; see the Submission Guidelines section of our website to learn how to submit samples for RT-PCR. Sample submission process begins only when you accept GSR Microarrays Policies. For each sample that you want to submit, fill in the requested information and click Add Sample. As you add samples, they will appear in the table near the top of the page. If you discover a mistake after adding a sample, click the Edit link beside the incorrect line in the table. After making your corrections, click the Update button in the box. If you wish to Cancel updating, you may do that as well, or Delete the sample altogether.
9 After all samples have been entered, scroll to the top of the page and fill in the Vanderbilt Billing Center box and VICC/DDRC Scholarship box. If you are a Vanderbilt investigator, Vanderbilt Billing Center is the billing center number that the work we do will be charged to. If you are using a scholarship or VICTR voucher, note the voucher number in the scholarship field. A valid billing center must accompany the scholarship or voucher for any charges not covered by the voucher. If you do not provide a correct center number, the order may be delayed or not processed, or GSR Microarrays will not be able to release your data. If you are external to Vanderbilt, in the Vanderbilt Billing Center box, enter the method of your payment ( Emory University, for instance). External investigators should contact us before submitting samples to receive a Quote for services. GSR Microarrays requires a Purchase Order prior to the release of data. Please make sure this information has been entered in your project before bringing or shipping samples to GSR Microarrays. The Special Instructions box should be filled in if there is any information we should know that does not fit elsewhere on the order form. Many of our commercial platforms are constantly expanding their Array platforms; new arrays not yet listed in our drop-down menus should be listed here. After entering all of the required information, click the Review Order button at the bottom of the screen. You will be shown a summary of the sample and billing data you just entered. Please review this information for accuracy, and click Place Order when you are through. Your samples are not entered into the system until you press the Place Order button. Once you submit your order, a confirmation will be mailed to all contacts on the Project, and the project PI. This contains a PDF copy of your order form and directions for physically submitting your sample. Make sure you read and follow these instructions; they contain important details such as how to label your tubes. If you do not follow these instructions precisely, GSR Microarrays cannot accept your samples. Requesting Hybridizations DNA Microarray Projects do not require a separate step for requesting hybridizations. All submitted DNA samples are to be hybridized unless the PI chooses not to hybridize based on QC/QA of the samples. Once the QC/QA of DNA samples has been posted and reviewed by the PI, the PI is asked to provide instruction regarding the specific samples that are/are not to be hybridized. As soon as your samples have been assessed by GSR Microarrays, they will be listed under Ready to Use and are eligible to be hybridized to a microarray. Start by clicking the Request New Hybridizations button on your project page. Enter payment information in the Billing Center field, just as you did when submitting samples. GSR Microarrays cannot hybridize your samples without valid billing information. The first time you request hybridizations for a gene expression project, you will be asked if you want to amplify your samples. This does not refer to the amplification step that is part of many standard protocols (i.e., Affymetrix IVT Express), but to a separate amplification with a Nugen kit. This is commonly used in experiments in which RNA is obtained at quantities below the minimum standards for the array manufacturer s protocol. Because this amplification can affect your final results, it must be performed on every sample in the project once one sample is amplified, they all must be. Please check with a GSR
10 Microarrays staff member before choosing to have your samples amplified. During bioanalysis, all of your samples were assigned an RNA Integrity Number between 0 and 10. GSR Microarrays recommends that only samples which scored a 7.0 or above be hybridized. If you wish to hybridize a sample that scored less than 7, click the Allow samples with an RNA Integrity Number less than 7.0 to be hyb d checkbox. Enter the Billing Center information, and the scholarship or VICTR voucher number, if you are using one, in the coupon field. A valid billing center must accompany the voucher for any charges not covered by the voucher in the Vanderbilt billing center field. If a center number is not given, the order may be delayed or not processed. Next, select the desired Microarray from the drop down, and select the samples you wish to hybridize from the list below. As in sample submission, any information or instructions that don t fit anywhere else should go in the Special Instructions. For example, if the particular array you want was not an option in the dropdown list, select Other and specify the array in the Special Instructions.
11 Click the Review Order button, verify that the summary is accurate, and click Place Order. The request for hybridization will now be placed in the queue for work orders in GSR Microarrays project management system. Viewing Results When your arrays have been hybridized and scanned, your results will appear on your project page. The exact format of your data will vary depending on the platform you chose, but raw, un-normalized data will always be posted (CEL, DAT, JPEG etc...). Additionally, as per PI request in the hybridization submission form regarding which samples need to be compared, our Bioinformaticians will perform the analysis accordingly and provide Normalized data along with Fold Change values. Descriptions of some of the common platforms data files are available on our web site under Data Analysis. To download your data, right-click on the file name and choose Save Target As. You can then specify a location on your hard drive and manipulate your data as you choose. We strongly recommend that you always back up your data to a safe place (such as DVD or CD) immediately. When you are ready to analyze your results, visit the Data Analysis section of our website. There, you will learn the fundamentals of data analysis, as well as what software and resources we make available to assist our clients. If you require further assistance, or would like us to perform the analysis, please contact a GSR Microarrays staff member to schedule a meeting with one of our bioinformaticists.
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