RealTime ready Custom RT-qPCR Assays and Panels Simple Online Configuration

Transcription

1 RealTime ready Custom RT-qPCR Assays and Panels Simple Online Configuration

2 Browse the Configurator at For life science research only. Not for use in diagnostic procedures. License Disclaimer: A license to perform the patented 5 Nuclease Process for research is obtained by the purchase of (i) both Licensed Probe and Authorized 5 Nuclease Core Kit, (ii) a Licensed 5 Nuclease Kit, or (iii) license rights from Applied Biosystems. This product contains Licensed Probe. Its purchase price includes a limited, non-transferable immunity from suit under U.S. Patent No. and 5,804,375 (claims -2 only (exp. 9/8/205)) and corresponding patent claims outside the United States. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research. The right to use this product in the 5 Nuclease Process under the corresponding patent claims outside the United States, can be obtained through purchase of an Authorized 5 Nuclease Core Kit. Except under separate license rights available from Applied Biosystems, no right under any other patent claim, or to perform commercial services of any kind, including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration, or to sublicense, repackage with other products, or resell in any form, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. ProbeLibrary is covered by US and other patent applications owned by Exiqon A/S. Locked Nucleic Acids (LNA) are covered by U.S. Patents No US 6,794,499 (exp. 9//208), US 6,670,46 (exp. 9//208), US 6,268,490 (exp. 3/9/208) & US 6,770,748 (exp. 3/9/208) and other patents and patent applications owned by Exiqon A/S and Prof. Takeshi Imanishi. The quenche Parts of the Software used for the LightCycler 480 System are licensed from Idaho Technology Inc., Salt Lake City, UT, USA. This product is covered by one or more of U.S. 6,97,520 (exp. 8/3/209), 6,303,305 (exp. 3/30/209), 6,387,62 (exp. 9/8/209), 6,503,720 (exp. 2/20/202), 6,730,50 (exp. 3/3/2022) and corresponding claims in their non-u.s. counterparts, owned by Roche Diagnostics GmbH and/or licensed from Idaho Technology, Inc. This LightCycler 480 Real-Time PCR System is a real-time thermal cycler licensed for research use only under U.S. Patent No. 6,84,934 (exp. 5/2/20) and corresponding claims in its non- U.S. counterparts, and under one or more of U.S. Patents Nos. 5,475,60 (exp. 2/2/203), 5,602,756 (exp. 3/4/20), 6,703,236 (exp. /29/200), 7,238,57 (exp./29/200), 7,504,24 (exp. /29/200), 7,537,377 (exp. 2/9/2020) or corresponding claims in their non-u.s. counterparts, owned by Applied Biosystems, LLC. No rights are conveyed expressly, by implication or estoppels to any other patent claims. The LightCycler 480 Real-Time PCR System is not intended for in vitro diagnostic use in the U.S. For further information on purchasing additional rights, contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California, 94404, USA. No right is conveyed expressly, by implication, or by estoppels under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5 nuclease methods. License rights to practice PCR methods under the foregoing Roche patents for research and other non-in vitro diagnostic applications may be purchased from Applied Biosystems or may be obtained by purchasing licensed reagents from Roche, Applied Biosystems, or other Authorized third party. For information on purchasing licenses for research and other non-in vitro diagnostic applications, contact the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. A license to practice PCR methods with real-time detection under patents of F. Hoffmann-La Roche Ltd, and Roche Molecular Systems ( Roche ) for in vitro diagnostic applications may be purchased from Roche or may be obtained by purchasing in vitro diagnostic reagents from Roche or any other Authorized third party. The LightCycler 480 Real-Time PCR System is not intended for in vitro diagnostic use in the U.S.This product is covered in-part by US 5,87,908 (exp. 2/6/204) or any foreign equivalents, co-exclusively licensed from Evotec OAI AG. The purchase price includes a license to practice the methods covered by US 5,87,908 (exp. 2/6/204) by using the product. Purchase of this product, however, does not convey to the purchaser a license or right to (i) commercially make, have made or sell reagents and/or kits, or (ii) buy or use reagents and/or kits provided by a third party used in conjunction with the product or any other thermocycler to practice the methods covered by US 5,87,908 (exp. 2/6/204) or any foreign equivalents. 2

3 How to Start 2 3. Launch the configurator 2. Selecting the Operating Mode The workflow selection window shows three options: Single Assays, Custom Panels, and Search Configurations Choose Assays RealTime ready Single Assays Search Assays Selecting Single Assays takes you directly to the Search Assays page. Choose your preferred search function by clicking tabs. 3

4 Search by Keyword This search function allows you to enter up to five search terms in any combination from any or all of the following five categories: gene symbol, gene name, public accession ID, biological process, or molecular function. Wildcard Search Use the asterisk character as a wildcard, or placeholder character, to produce a more general search. When using, for example CCN*, you will find all gene names beginning with the keyword CCN. Note that wildcard searching is disabled by default. To activate wildcard searching, uncheck this box. Search Restrictions To speed up your search, you can limit the search to different search areas. 2 AND or OR Functions When you want to search assays for a gene containing all the keywords, type AND in full capitals between each search term (e.g., CCND AND cell cycle ). When you want to perform a broader search for all genes that contain any of the keywords, type OR in full capitals between the search terms (e.g., CCND OR cell cycle ). 4

5 Choose Organism Next, choose the organism for which your assays will be designed. You can choose to get an assay for all organisms, or choose one or two by checking the appropriate boxes. Assays for Other Organisms can be designed using the Universal ProbeLibrary Assay Design Center. UniRT mirna assays from Exiqon matching your search term can also be searched. Start your search by clicking the Search button Search by Focus Panel The Search by Focus Panel function allows you to search for your assays in pre-selected targets from different biological pathways or gene superfamilies ranging from Apoptosis to WNT Signaling. The panels are also grouped into categories, such as Oncology or Signal Transduction. Click on a magnifier symbol to view the selection for the respective organism and select relevant assays. 5

6 Batch Upload The Batch Upload function allows you to upload a text file that specifies your targets in the form of accession ID s from the listed fields, or RealTime ready Assay IDs (check at least one field). Before you upload your text file, make sure that each search term is separated with a carriage return. Do not combine terms on a single line or use punctuation to separate terms. 6

7 Search by Pathway The Search by Pathway function allows you to select your targets from a variety of interactive maps of cellular or signaling pathways. Click on a target to add it to your search. A small window confirms the selection. To start the search for all targets contained in the search field, choose the correct organism and click Search

8 2. Choose Assays Depending on the type of search you have selected, you will get a list of assays matched to your search terms and selected organisms. The list gives you a comprehensive overview of design information for the individual assays. If no matching Catalog Assay is available, the result is marked with tbd, which means that an appropriate assay will be designed and tested for your selected target. Clicking on the Transcript leads directly to the Ensembl database. Clicking on Details or the Assay ID 3 brings you to the Assay Details page, which provides a graphical display of the location on the transcript, as well as a list of all isoforms or accession IDs that can be detected with this particular assay. 8

9 Select the desired assays using the check boxes and one of the Add to List buttons. The My Assays window will be updated to show the number of Catalog or Designer Assays you have on your list. To edit and review your current selections, click the Edit List button. 3. Edit List The Edit List window shows your current assay selections. Moving the mouse cursor over an assay will pop up a box listing brief details for the assay. Use the buttons on the right to edit, refine, or print your search results. Once you have decided which assays to purchase, move on to the Review page

10 4. Review your Selection The Review window displays a summary of your selections. To make changes to your selections, click Edit List to return to your list of assays. If you are satisfied with your selections, click Continue to obtain a Configuration Number for your assay; this number is required for ordering. Your configuration(s) are not complete if no Configuration Number is assigned. If you close your browser, your configurations will not be saved. Your configurations will be assigned a unique Configuration Number, which is mandatory for ordering an assay, when you click "Finalize". 0

11 5. Order Your Custom Assays For ordering these configurations please follow the instructions in the that you will receive after you click the "Finalize" button.

12 RealTime ready Custom Panels The Custom Panels mode allows you to configure RealTime ready Custom Panels with assays of your choice for human, mouse, and rat genes on LightCycler 480 Multiwell Plates, 96 or 384. Se. Select Layout and Assign Name arh and 5 Several different layouts are provided for your custom panel on LightCycler 480 Multiwell Plates. Download an overview of all plate layouts or click on the View Layout icon to select the one suitable for your custom panel. Find a description of the controls and reference genes provided for each custom panel by clicking the information icons. You can choose between layouts containing 2 or 3 selectable reference gene assays at defined positions (e.g., RealTime ready Custom Panel 96-6) or a layout that contains control assays for the RT step and for DNA 2

13 contamination (e.g., RealTime ready Custom Panel 96-6+). An overview of all panel layouts and a description of the Error Detection Feature can be found on pages Once you have decided on a layout, you must provide a unique name for your panel. Click Configure to start the configuration process Configuration Overview This page gives you a short overview of the configuration workflow, providing links to the respective sites. The My Panel window will accompany you throughout the configuration process. It provides you with real-time information about the panel you are currently configuring. The window shows the number of assigned wells, empty wells, controls, and reference genes. 3. Search and Choose Assays This overview briefly explains the four search functions. Choosing a search function will take you to the assay search and selection process, as described on pages

14 Search Result Page On the Search Result page, links to detailed descriptions of the assays are provided when you click on the Assay ID or on the Assay Details button. Add the desired assays to your panel by using the check boxes and one of the Add to Panel buttons. You can view and edit your selection, clicking Edit Panel. Edit Panel The Edit Your Configuration page lists your current panel content. Move the mouse cursor over the assays to pop up a window listing brief details for the respective assay. Before you move on to the Review page, select your preferred reference genes for the predetermined location on your panel. 4. Select Reference Genes On the Choose Reference Genes page, choose the organism, and then select assays from the result list. Note that all reference gene assays need to be from the same organism. The My Panel box indicates the number of reference genes you must select for your layout. If you have selected a layout with controls, the control assays for the same organism will be added to your panel automatically. 2 4

15 3 4 5 Use the View Panel button to obtain a preview of your custom panel. You can also print the contents of your panel as a list for your records. When you are satisfied with your selection of assays, move on to the Review page. 5

16 2 5. Configuration Review To make changes to your selections, click Edit Panel to return to your list of assays. If you are satisfied with your selections, click Continue to obtain a Configuration Number for your assay; this number is required for ordering. 6

17 Your configuration(s) are not complete if no Configuration Number is assigned. If you close your browser, your configurations will not be saved. Your configurations will be assigned a unique Configuration Number, which is mandatory for ordering an assay, when you click "Finalize". 6. Order your Custom Panel For ordering these configurations please follow the instructions in the that you will receive after you click the "Finalize" button. 2 3 My Configurations Your work space, called My Configurations, is accessible from most Configurator windows. By default, all your configurations and assays are automatically stored as not finalized, meaning that you can freely edit and modify them, as well as export them as text files. To continue working on a configuration, click on Edit Panel to go to the Configuration page, where you can access all other functions. Click on Add to List next to an un-finalized assay to go directly to the Edit List page. Finalized panels and assays have a configuration number assigned to them and cannot be edited or deleted. Finalized panels can, however, be copied and renamed; the duplicate panel can then be edited under the new name. Your original configuration will still be saved under its original name and configuration number. 7

18 You can return at a later time, and access all your work by using the Search Configurations page. On this page, you can search, view and download the relevant files associated to the previously ordered configurations. You can browse the Configurator at

19 RealTime ready Custom Panel Layouts LightCycler 480 Multiwell Plate 96, white Reference Genes (required) For each layout, fixed positions are reserved for the respective number of reference genes. You can select reference gene assays specific for the species on your panel from a list of 9 different targets. Controls (optional) 9

20 RT+ controls for initial RNA quality and reverse transcription reaction: 3 assays, each targeting different positions of the same transcript: 3 end, center region, and 5 end. RT- control detects residual DNA: 2 identical assays are amplified with a) cdna sample b) RNA sample LightCycler 480 Multiwell Plate 384, white Trademarks: LIGHTCYCLER, REALTIME READY, and NIMBLEGEN are trademarks of Roche. Exiqon, LNA ProbeFinder and ProbeLibrary are registered trademarks of Exiqon A/S, Vedbaek, Denmark. 207 Roche Diagnostics. All rights reserved. 20