TABLE OF CONTENTS PI REGISTRATION

Transcription

1 Microarray Core Facility An Introduction & User Guide to the MCF s Web-Based Ordering & Sample Tracking System TABLE OF CONTENTS PI REGISTRATION...2 User ID Set-Up...2 Changing the Password...3 Adding Lab Members...3 PI FEATURES...4 Search Requests...4 View Billing Information...4 Modifying Account Information...4 SUBMITTING REQUESTS...5 Submitting a New Request (general)...5 SAQC (Sample Quality Check)...5 Entering a new SAQC Request...6 Sample Information Requested...6 Entering Your Sample Information...6 Viewing the MAID SAQC Results...8 cdna REQUESTS...9 To Begin: Entering a cdna Request...9 cdna Processing Available at the MCF...10 Selecting the Samples...11 Completing the cdna Request: Experimental Design Considerations...11 AFFY REQUESTS...12 To Begin: Entering an Affymetrix Request...12 Affymetrix Processing Available at the MCF...13 Selecting the Samples...13 Completing an AFFY Request...14 CLONE PICKING REQUESTS...14 Entering a Clone Picking Request...14 Information to Include in a Clone Picking Request...15 Dropping off Plates

2 Welcome to MAID! (MicroArray Information Database). MAID is the web-based ordering and sample tracking system for Baylor College of Medicine Microarray Core Facility (MCF) users. Any Baylor College of Medicine researcher can submit requests to the Microarray Core Facility using MAID. MAID was co-developed by the MCF at Baylor College of Medicine and Stone Bond Technologies in an attempt to simplify the complex process of making requests and submitting samples to the facility. The MCF hopes this will help researchers at Baylor College of Medicine keep better track of their samples, monitor sample progress and manage the data generated through the MCF. Access to MAID is limited to those users within the Baylor network. Those labs accessing MAID from outside of the BCM campus will have to download the VPN (Virtual Private Network) software in order to login. NOTE: At the current time, MAID is only supported using Microsoft Internet Explorer 5.0 and higher for PC users and Netscape 7.0 and higher for Mac users. As Netscape will soon be discontinued, we are working to expand its usability for Safari and/or FireFox. Well, let s get started. PI REGISTRATION In order to begin using MAID, each person will need a username and password. Each Private Investigator (PI) will need to first fill out a Registration form and fax it to the Core ( ). This form contains basic information about the PI and which charge sources he/she wants their lab members to have access to for billing requests. The form is available on the MCF website and by fax or . If you have trouble finding it or do not have access to the BCM intranet please contact the MCF at or microarray@bcm.edu. NOTE: Please make sure to fill out the form COMPLETELY! User ID Set-Up The preferred User ID (Baylor ID) is the BAYLOR ENTERPRISE username. This is the username we will assign you to logon to MAID. Any very memorable 5 character name will do if you object to the use of your enterprise address but please realize there may be delays in inputting requests into the MAID system if you require resetting of your User ID or password. The User ID should not be the 6 digit Baylor ID badge number. The MCF also needs to keep records of which types of accounts/grants are being billed at the Core, so please make sure to put the SAP account number AS WELL AS what type of account it is. NIH does not count!! Please indicate a more detailed account type (i.e. NIDDK, NCI, American Heart Association, etc.) Once the PI is registered, he/she will receive an notification along with their username and password. The PI should login on the MAID system ( and immediately change their password to something they will remember. 2

3 Changing the Password Changing the password can be easily accomplished by clicking on the name to the far right of the screen once logged in. Next click the checkbox at the bottom of the page to Change Password. Enter a password that is easily remembered at your earliest convenience. Resetting a forgotten password will delay inputting your request. Adding Lab Members The only thing remaining for the PI to do is to add lab members (LM) to their lab. The LMs will have access to the accounts listed under the Accounts tab. To add a LM, scroll over the User Info tab and select Add. Again, for simplicity have their username be their Baylor username, along with a default password. Please inform them to change their password when they login. NOTE! It is the responsibility of the PI to add lab members to their MAID account. The MCF is not responsible for adding members to the lab. Remember, this should not be everyone in the lab, just those members who use the MCF to submit requests or who will be asked to drop off samples. The lab members will have to choose an account number when in order to make a request, so make sure they know which account(s) to use. Figure 1: Adding a Lab Member (LM) to a PI s lab Once you have added all of the LM s, let them know so they can do all the hard work of submitting requests. Each LM needs to follow the first step as well. They should logon to MAID and change their password. Clicking the name to the far right of the screen once logged in. Then click the check-box at the bottom of the page to Change Password. 3

4 PI FEATURES In addition to submitting their own requests, PIs can view any or all of the requests made by their LMs. Search Requests Requests can be searched by scrolling over Request and selecting Search. The search can be by requester, request ID, request type, etc. A list of requests will be shown based on the search criteria. Simply click one of the hyperlinks on the list to view details about a request. View Billing Information PIs can also view the billing in their lab to view what has services have been billed to their lab. Scroll over Billing and select the search parameters, or just click Search and an Excel spreadsheet can be opened to view all of the requests that have been billed to the individual account(s). Figure 2: Search Billing (PI) Modifying Account Information By scrolling over Accounts, PIs can add or delete the account numbers for billing requests to their lab. This is an option as a PI but we recommend that you permit the MCF MAID administrators to delete any accounts you wish to remove. NOTE: If you delete an account number that has been used for any past requests, those requests will no longer show up in the MAID system, so please be careful!! If an account needs to be removed, please inform the MCF and we will take care of the deletion. Finally, PIs can generate reports by defining several different parameters. PIs can search by Request Type, Request Status, and even the type of account. Click Search and the next page will display a table that can easily be copied into an Excel spreadsheet. 4 Figure 3: Generating Reports (PIs)

5 SUBMITTING REQUESTS Now that all the administrative duties are completed, requests can be submitted to the MCF. The current services are as follows: SAQC (Sample Quality Check) AFFY (Affymetrix GeneChip Services) CDNA (cdna Array Services which include all microarray platforms but Affymetrix ) CLON (Clone Picking Service), and DATA (Data Analysis Services) Submitting a New Request (general) Scroll over Request and select New from the menu. Notice that next to cdna and Affymetrix there is a qualifier ( Needs completed SAQC request ). Users must have a completed SAQC request in order to submit any samples for cdna or Affymetrix services. The MCF will only accept and process samples that have been quality checked and passed for use on array platforms. Figure 4: Selecting a New Request SAQC (Sample Quality Check) One of the most important factors in doing any microarray experiment is the quality of the starting material. The starting sample for any array experiment, either total or polya+ RNA, must be extremely pure with little to no degradation. The MCF uses two instruments to test the quality of the samples, the Agilent 2100 Bioanalyzer (Agilent Technologies) and the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies; see website for details). 5

6 Entering a new SAQC Request Click on Sample Quality Check to start a new SAQC request. The user name and user s PI information are automatically displayed and cannot be edited. There are four drop-down menus that must be selected. 1. The first indicates what charge source the request should be billed to. 2. Second, select the type of report file to be used to display the results. Choices are Printed, Electronic ( ) or both. 3. Third, select whether the MCF should save the samples and return or to discard them. Samples left longer than 2 weeks from request completion will be thrown away, regardless. 4. Fourth, you need to choose how many samples you want processed through SAQC. There is an 11 sample limit on how many samples you can enter per request. 5. Finally, you need to indicate whether the sample should be processed as Pico or Nano depending on the concentration cut-offs outlined in the MAID form and on the MCF website. Please keep in mind that, unless the user indicates otherwise in the notes, samples chosen for the Nano chip will be assessed according to the Affymetrix One-Cycle protocol concentration. And samples chosen for the Pico chip will be assessed according to the Affymetrix Two- Cycle protocol concentration. NOTE: The Agilent Bioanalyzer 2100 Data Review Software must be installed in order to receive and open the electronic file for each request. There are download instructions on the MCF website, or you can contact Agilent directly at genomics@agilent.com. Enter the serial number DE when prompted. Sample Information Requested In order to aid the researcher in complying with MIAME (Minimum Information About a Microarray Experiment) standards (which is necessary if the intent is to publish), biological information about each sample is recorded and will be attached electronically to any and all data obtained in the Core. Entering Your Sample Information Fill in the required information for each sample: Sample Name- Each sample will be given a unique MAID Sample ID Number. The MAID Sample ID Number is the identifier that should be used on 1.5mL tubes when you submit samples for SAQC, not your sample name. You will need to go back into MAID to determine these numbers after you receive the MAID confirmation informing you of successful submission of a SAQC request. Est. Conc. (ηg/µl)- There is a concentration limit on the Bioanalyzer. For a Nano chip we require the user to dilute their samples to a range of ng/µl. For a Pico chip the concentration range is pico-grams but we request that users submit undiluted pico RNA for the SAQC analysis. Fill in the estimated concentration of the stock RNA sample before any dilution occurs. 6

7 Dilution Factor- The dilution factor (if any) for the submitted samples; if the samples were not diluted enter 1:1 (NOT 1:0). This information is used to calculate the actual concentration of the samples. The dilution factor used to prepare the RNA within the parameters for the Agilent BioAnalyzer should be entered here and will be used by MAID to calculate the estimated concentration of the RNA you actually submit for SAQC analysis. This number will later be used to calculate your ACTUAL RNA concentration based on the NanoDrop reading recorded at the MCF. Sample Type- Choose the type of sample from the drop down menu. If your sample type is not there please contact the MCF and we can add it. Sample Info- Click the link to open the new window Sample Information The Sample Info link opens a new window for the user to enter information about each sample submitted. Figure 5: Sample Info window NOTE: THIS IS ENTIRELY FOR THE USER S BENEFIT! The information provided will describe such things as species type, sex, tissue, treatment, as well as other information that will be valuable and necessary in order to publish the microarray data. Please take this seriously, it may take a while, but it will be worth the time and effort in the long run. Click Save to save the sample info and return to the main request page. 7

8 As a default, the next sample info window will contain the exact same info as the previous. That is so you will not have to go through the trouble of reentering all the same information for every single sample, only change what needs to be changes (treatment, food source, etc.) When all the necessary information has been entered, be sure to read and check the disclaimer box at the bottom before clicking Submit. Figure 6: Check the disclaimer The request has now been submitted to the MAID system. But don t forget, the samples must be delivered to the MCF. We do not pick up samples. In case of forgetfulness, an automated will be sent with notification that the request was received and a reminder to deliver the samples to the MCF at the DESIGNATED TIMES. The Core is very busy, so we ask users to bring their samples at the specific times that we have designated for receiving samples. YOU MUST SUBMIT A REQUEST BEFORE DROPPING OFF THE SAMPLES!! The MCF will not accept samples unless there is a request already in the system. It only takes a few minutes, so please fill out the request before you bring the samples. That is really it. Sit back, relax, and wait for the samples to be checked. When the request is completed, another will be sent informing you that your project has been completed. You will need to log back into MAID to view the results of your sample QC. Again, the Agilent Bioanalyzer Data Review Software is required in order to open the.xad files, which are located under the heading Data Files. Be sure you check all of the comments from the MCF technician. The comments for each sample and the Notes contain important information about each sample. Viewing the MAID SAQC Results You will be instructed in the you receive upon completion to log back into the MAID system to view your results. Each lab member may view the results of all projects submitted by that LM, and each PI can see all projects submitted by each LM. Log into MAID and select the option for New Request. 8

9 Once you have opened the SAQC request, scroll down to the end of the page to get information on: The actual concentration of the samples Any notes about the specific samples from the MCF technician Whether or not the sample passed SAQC Links to open the Agilent.xad data file(s) and the NanoDrop file(s). The Agilent.xad file is obtained by downloading the link under the Data File field. There may be more than one file so make sure you look carefully. The technician will generally indicate in the Notes when there are multiple files for your project. The NanoDrop file is currently located under the Genomic DNA Gel Image field. There may be more than one file here, as well, so please do check. This file is in Excel format and can be viewed without any special software. If and when you decide to have the MCF perform any array services, you will be able to select any of the Passed or Passed With Reservation samples for AFFY or cdna requests. cdna and AFFY requests are very similar to fill out. For both, you may only select samples that have passed SAQC for a request. For information about the sample sign-off procedure for using any Failed samples, please contact the MCF (microarray@bcm.edu). cdna REQUESTS Before Starting cdna: All samples must pass SAQC (Agilent AND Nanodrop ) quality control. The samples must meet minimum requirements for quality control: RIN of 8.5 or higher, 18/28S ratio of 1.6 or higher, 260/230 or 1.5 or higher, intact 18S and 28S peaks (for eukaryotic samples), and low baseline. NanoDrop must demonstrate sufficient concentration of starting material which is variable depending on desired platform and protocol. Please contact the MCF to arrange an experimental design session to choose the best platform for your experiment. When these samples pass SAQC, they will be available to select for cdna requests. Until the sample passes SAQC they WILL NOT be available for selection in an array platform request.. To Begin: Entering a cdna Request To enter a cdna request, begin by logging onto MAID. Scroll over Request and select New. Click cdna to fill out a new request. Again, the requestor and PI information will be displayed automatically in a noneditable form. Next, select the appropriate account number (charge source) for this request. Figure 7: New cdna Request 9

10 The next section of the request itemizes the different cdna Array services the MCF provides. Click the drop-down menu beside each one and choose the particular services you would like performed. cdna Processing Available at the MCF Amplification- If you do not have the following minimum cut-offs, you will need amplification if it is available for your particular protocol: - Agilent: Expression: 1ug total - Agilent acgh: 1.0µg 20.2 ul - Agilent microrna: 25.0ηg/uL - NimbleGen Expression: 1000ng/uL. If you re protocol isn t listed, please discuss with the MCF director or lab manager via microarray@bcm.edu. If you have these minimum requirements and your samples have passed SAQC, please choose "NONE since you will not require any amplification. Labeling if you are performing your own sample labeling step, please choose "NONE" If amplification was chosen above, "Amino-Ally (arna) Labeling. If the sample is total RNA and does not need to be amplified, choose Amino-Allyl (Total RNA) Labeling If the sample is for acgh or ChIP-on-Chip and is therefore DNA, select Amino-Allyl (DNA) Labeling. Hybridization- - if the MCF will be performing the hybridization, choose "GeneTAC HybStation, otherwise, select "NONE." We will use the Agilent Hybridization Chambers in the Agilent Hybridization oven for all Agilent samples and the MAUI Hybridization Station for all NimbleGen samples. Laser Scanning- if the MCF is scanning your arrays, select either ScanArray 5000 or GeneTAC UC-4. We will actually scan all Agilent and NimbleGen slides on the Agilent Scanner which is not yet an option in the menu. If you have your own scanner and wish to scan your own chips, please select "NONE. Chip Type- please select the appropriate chip type for the request. If you have purchased or will be supplying your own chip(s), please select "NONE SELECTED and identify the chips in the Notes section of the request. Please designate if you'd like for us to discard the samples or keep them for you to collect. Please keep in mind that we discard all unclaimed samples after 2 weeks. Select the samples from the sample list that have passed SAQC. 10

11 Selecting the Samples The selection is made by checking the box to the left of the sample name and clicking on the "Select" button at the bottom of the page. Alternatively, samples can be searched by sample name, Request ID or date of submission to SAQC. After samples have been selected, enter the volume amount that will be submitted. If the volume and concentration do not meet the standards for the Amino-Allyl Labeling protocol selected, the request will not be allowed. Figure 8: Selecting samples from the Sample Info Link Completing the cdna Request: Experimental Design Considerations Experimental design and analysis methodology are important decisions you must make before beginning an experiment. If you have any questions about which experimental design is appropriate (Reference or Direct), please contact the MCF at Enter any Notes in the Notes section if you have any special requirements for the request. Finally, read the disclaimer notice at the bottom and indicate that you've read and understood by clicking the box to the left of the statement. You must click the "Submit" button at the bottom of the page to complete the request. After completing the request, you will receive a confirmation along with the times to drop off the samples to the MCF. Figure 9: Completing a cdna Request 11

12 AFFY REQUESTS Before Starting AFFY: All samples must pass SAQC (Agilent AND Nanodrop.) meaning the samples must meet minimum requirements for quality control: RIN of 8.5 or higher, 18/28S ratio of 1.6 or higher, 260/230 or 1.5 or higher, intact 18S and 28S peaks (for eukaryotic samples), and low baseline. Nanodrop must demonstrate sufficient concentration of starting material for Affymetrix or the Affymetrix -compatible amplification kit you plan to use. You must discuss alternatives with the MCF before the request can be entered. When these samples pass SAQC, they will be available to select for AFFY requests. Until the sample pass SAQC they WILL NOT be available for selection in an array platform request. To Begin: Entering an Affymetrix Request To enter an Affymetrix request, begin by logging into MAID. Scroll over Request and select New. Click AFFY to fill out a new request. Again, the requestor and PI information will be displayed automatically in a non-editable form. Next, select the appropriate account number (charge source) for this request. Figure 10: Submitting a New AFFY Request 12

13 The next section of the request itemizes the different Affymetrix services provided by the MCF. Click the drop-down menu beside each service and choose the particular services necessary for completion of your project. Affymetrix Processing Available at the MCF Amplification- unless you are doing a small sample preparation (and have already had an experimental design meeting with Dr. White), please chose "NONE. Labeling If you are doing your own labeling, please choose "NONE If you are doing small sample prep (from Affymetrix please chose "Small Sample Prep Labeling" or an alternative amplification method), If the sample does not require additional amplification, choose Affymetrix Labeling. NOTE: If amplification is selected the user must select small sample prep labeling. Hybridization- if the MCF will be performing the hybridization, choose "Affymetrix Hybridization"; otherwise, chose "NONE." Washing/Staining- if the MCF will be performing the washing/staining step, choose "Affymetrix Washing/Staining"; otherwise select "NONE. Laser Scanning- if the MCF will be scanning the GeneChips, select GeneChip Scanner 3000 ; otherwise select "NONE. Chip Type- please select the appropriate chip type for the request. If you have your own chip(s), please select NONE and indicate the type of arrays you will be providing in the Notes section of the MAID request. You should be prepared to drop off your array(s) along with your sample(s) at the designated sample drop-off time. Please indicate whether the MCF should discard the samples or keep them for you to collect. Please keep in mind that we discard all unclaimed Figure 11: Selecting samples from the Sample Info Link samples after 2 weeks. Selecting the Samples Select the samples from the sample list that have passed SAQC. The selection is made by checking the box to the left of the sample name and clicking on the "Select" button at the bottom of the page. Remember, only samples that have passed SAQC are available to be selected. 13

14 Next, enter the volume amount to be submitting. If the volume and concentration do not meet Affymetrix standards, you will not be allowed to submit the request. Completing an AFFY Request Please enter any additional requirements for the request in the Notes section at the bottom. Finally, read the disclaimer notice at the bottom and indicate that you've read and understood by clicking the box to the left of the statement. You must click the "Submit" button at the bottom of the page to complete the request. After completing the request, you will receive a confirmation along with the appropriate times to drop off the samples to the MCF. Figure 12: Completing an AFFY Request CLONE PICKING REQUESTS This is a simple request for users wishing to obtain cdna clones of interest from the MCF, either for their own experiments or to verify gene expression results from microarray experiments. Information needed to request clone picking: Target ID Clone ID Accession # (this information is available from our clone lists on the MCF website). Once this information is obtained by searching our Mouse and/or Human clone list(s), the user may logon to MAID and enter the clone picking request. Entering a Clone Picking Request To enter a clone picking request, click on Request, then New. This will open a new web page. On this page please click on Clone Picking. This will take you to the CLON (clone picking) request form. 14

15 Information to Include in a Clone Picking Request Please fill out this form completely. Also, include the Target ID, Clone ID and Accession # of each clone requested. Click on the submit button. Figure 13: Filling out a CLON (Clone Picking) Request After submitting the form, an will be sent indicating that the user needs to bring LB/Amp plates to the Microarray Core Facility (MCF). Make sure to bring at least one more plate (more if more than 5 clones are requested) than the number of clones you are requesting. For example, if you would like to purchase 10 clones, then bring 12 LB/Amp plates. We will return any unused plates upon request. Dropping off Plates Once the MCF has received the request and you have gotten an stating that your request has been received you will need to drop off the LB/Amp plates during normal sample drop-off times. Once the MCF has received the LB/Amp plates, an MCF technician will streak the requested clone onto the LB/Amp plates provided and grow them overnight. Picking up Your Clones Once the request is completed, the MCF will send another indicating completion of the request. At this time you may make arrangements with the technician who completed your request to come to the MCF to pick up the clones by ing the technician directly or calling and asking to speak with that technician. 15