nCounter® Gene Expression Assay
Contents
1.2. FIGURE 1 2 Suggested workflow for the nCounter Expression Assay Manual Processing Hands on Time Dayi Set Up Hybridization 5 minutes Automated Processing Hands on Time Day 2 Set Up Prep Station Run 5 minutes Set Up Data Collection 5 minutes Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation 7 nCounter Gene Expression Assay Materials Required The following tables list the recommended materials and instrumentation required to run the nCounter Gene Expression Assay TABLE 1 1 Materials required for Total RNA Standard Protocol nCounter Gene Expression GX CodeSet NanoString Technologies GXA P1CS XXX OR nCounter Plex Expression Assay Kit GXA 2PLX XXX or GXA 4PLX XXX nCounter Master Kit GX only NanoString Technologies NAA AKIT XXX QIAGEN RNeasy Kit or similar QIAGEN 74104 or 74106 Disposable gloves various Total RNA 100ng or 150ng per hybridization assay If total RNA has been isolated by some other method please contact NanoString customer support support nanostring com We highly recommend verifying the integrity of your total RNA samples via denaturing PAGE or Bioanalyzer Agilent Technologies before proceeding with hybridization TABLE 1 2 Materials required for Cell Lysate Protocol nCounter Gene Expression GX CodeSet NanoString Technologies GXA P1CS XXX OR nCounter Plex Expression Assay Kit GXA 2PLX XXX or GXA 4PLX XXX nCounter
3. b Cell lysates should be aliquoted and stored at 80 C Avoid freeze thaw cycles 3 Remove aliquots of both Reporter CodeSet and Capture ProbeSet reagent from the freezer and thaw on ice Invert several times to mix well and spin down reagent i IMPORTANT After it has thawed inspect the tube of Reporter CodeSet to make sure no colored precipitate is present If you see a colored precipitate heat the entire tube to 75 C for 10 minutes and cool at room temperature before using 4 Labela provided 12 tube strip and cut it in half so it will fit in a picofuge 5 Add 10uL of Reporter CodeSet reagent to each tube Store remaining Reporter CodeSet at 4 C for up to 1 month 6 Add 10uL of hybridization buffer to each tube 7 Add sample according to your protocol type as follows a If following the Total RNA Standard Protocol Add total RNA sample maximum volume 5uL for a total of 100ng to each tube Go to Step 8 b If following the Cell Lysate Protocol Add cell lysate sample maximum volume 4 L for a total of approximately 10 000 cells per hybridization assay Using less than 10 000 cells reaction will result in fewer counts gene c If using attenuation mix es add luL of each mix 8 Add RNAse free water to each tube to bring the volume of each assay to 25uL 9 Pre heat thermocycler to 65 C Program the thermocycler using 30L volume calculated temperature heated lid and forever time setting Do not set the thermocycle
4. be more variable in a hybridization oven To use a hybridization oven place a large beaker full of water in the oven to ensure a humid environment Do not place the samples in the water beaker evaporative loss causes the water temperature to be below the air temperature in the oven Place the samples in a dry rack in the middle of the oven shelf or tape the strip tubes to a rotator in the center of the oven Make sure that the strip tubes and or rack do not touch the sides or bottom of the oven Failure to follow these instructions may result in uneven hybridization temperatures which can compromise the results 10 Add 5uL of Capture ProbeSet to each tube immediately before placing at 65 C Cap tubes and mix the reagents by inverting the strip tubes several times and flicking with your finger to ensure complete mixing Briefly spin down and immediately place the strip tube in the 65 C thermocycler Minimizing the time between the addition of the Capture ProbeSet and the placement of the reaction at 65 C will increase the sensitivity of your assay 11 Incubate hybridization assays for at least 12 hours Hybridizations should be left at 65 C until ready for processing Maximum hybridization time should not exceed 30 hours 12 Once removed from the thermocycler proceed immediately to post hybridization processing with the nCounter Prep station Do not store hybridizations at 4 C Molecules That Count Translational Research Gene Express
5. sublicensable research use only license to use the proprietary nCounter Analysis System only in accordance with the manual and other written instructions provided by NanoString Except as expressly set forth in the terms and conditions no right or license whether express implied or statutory is granted by NanoString under any intellectual property right owned by or licensed to NanoString by virtue of the supply of the proprietary nCounter Analysis System Without limiting the foregoing no right or license whether express implied or statutory is granted by NanoString to use the nCounter Analysis System with any third party product not supplied or licensed to you by NanoString or recommended for use by NanoString in a manual or other written instruction provided by NanoString Trademarks NanoString NanoString Technologies nCounter Molecules That Count nSolver Plex2 ChiP String miRGE and nDesign are registered trademarks or trademarks of NanoString Technologies Inc NanoString in the United States and or other countries All other trademarks and or service marks not owned by NanoString that appear in this document are the property of their respective owners Copyright 2008 2013 NanoString Technologies Inc All rights reserved nanoString NanoString Technologies Preface Conventions Used Note Types Fonts Procedures Contact Information Chapter 1 Ptro duc
6. Master Kit NanoString Technologies NAA AKIT XXX RLT buffer or similar QIAGEN 79216 Disposable gloves various Cell lysate 2 500 15 000 cells per microliter QIAGEN buffers and a QIAGEN cell lysate procedures with modifications outlined in Chapter 2 have been tested internally at NanoString Technologies If using a cell lysis procedure other than QIAGEN S please contact NanoString customer support support nanostring com for additional information nanoString 8 3 d NanoString Technologies USER MANUAL TABLE 1 3 Instruments required for Total RNA Standard Protocol and Cell Lysate Protocol NanoDrop ND 1000 Bioanalyzer 2100 Pipette for 0 5 10yL Pipette for 2 20yL Pipette for 20 200yL Tube Strip Picofuge DNA engine thermocycler hybridization oven or similar nCounter Prep Station nCounter Digital Analyzer Memory stick NanoDrop Technologies Agilent Rainin Rainin Rainin Stratagene MJ Research BioRad NanoString Technologies NanoString Technologies various n a G2940CA L 10 L 20 L 200 400540 PTC 200G PTC 1148 PTC 0220Gt PTC 0221Gt PTC 0240Gt NCT PREP 120 NCT DIGA 120 n a nCounter system performance data was generated on model PCT DNA engines Other instruments may produce non standard results when used with the nCounter assay Any one of these instruments will meet the requirements of the nCounter Assay Contact Information NanoS
7. Qiagen recommendations see Qiagen RNeasy Mini Handbook supplied with product numbers 74104 and 74106 with the following modifications a Cells should be lysed at concentration between 3 500 and 15 000 cells uL of RLT buffer The nCounter Plex cell lysate hybridization procedure has been optimized for 15 000 mammalian cells reaction or the equivalent of approximately 150ng of total RNA b Cell lysates should be aliquoted and stored at 80 C Avoid freeze thaw cycles Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation 15 nCounter Gene Expression Assay 3 Remove aliquots of both Reporter CodeSet and Capture ProbeSet reagent from the freezer and thaw on ice Invert several times to mix well and spin down reagent i IMPORTANT After it has thawed inspect the tube of Reporter CodeSet to make sure no colored precipitate is present If you see a colored precipitate heat the entire tube to 75 C for 10 minutes and cool at room temperature before using 4 Create a master mix for each sub CodeSet by adding 130uL of hybridization buffer to the tube of Reporter Probes for each CodeSet Do not remove the Reporter Probes from tube RNAse free water may also be added to this mix if the volume of the individual RNA samples is less than 5uL and is constant Add enough water for 13 assays to allow one assay s worth of dead volume Do not add the Capture ProbeSet to the master mix Inve
8. e used The use of a thermocycler is recommended if possible Due to less stringent temperature control assay results may be more variable in a hybridization oven To use a hybridization oven place a large beaker full of water in the oven to ensure a humid environment Do not place the samples in the water beaker evaporative loss causes the water temperature to be below the air temperature in the oven Place the samples in a dry rack in the middle of the oven shelf or tape the strip tubes to a rotator in the center of the oven Make sure that the strip tubes and or rack do not touch the sides or bottom of the oven Failure to follow these instructions may result in uneven hybridization temperatures which can compromise the results 10 Add 5uL of Capture ProbeSet to each tube immediately before placing at 65 C Cap tubes and mix the reagents by inverting the strip tubes several times and flicking with your finger to ensure complete mixing Briefly spin down and immediately place the strip tube in the 65 C thermocycler Minimizing the time between the addition of the Capture ProbeSet and the placement of the reaction at 65 C will increase the sensitivity of your assay 11 Incubate hybridization assays for at least 12 hours Hybridizations should be left at 65 C until ready for processing Maximum hybridization time should not exceed 30 hours nanoString 16 i p s NanoString Technologies USER MANUAL 12 Remove tubes from the t
9. ex or pipet vigorously to mix as it may shear the Reporter Probes Mixing should be done by flicking or inverting the tubes Also if you use a microfuge to spin down tubes do not spin any faster than 1 000 rpm for more than 30 seconds and do not pulse it to spin because that will cause the centrifuge to go to maximum speed and you may spin your CodeSet out of solution i IMPORTANT To facilitate downstream analysis in the nSolver Analysis System the same sample Calibration sample must be run across both CodeSets in the same lane at least once per study see Figure 2 1 This means that you must hybridize the same Calibration sample in both sub CodeSets one time For more information on the Calibration sample see the nCounter Expression Data Analysis Guidelines FIGURE 2 1 Hybridization setup To facilitate downstream analysis in the nSolver Analysis System a Calibration sample must be assayed across both Sub CodeSets in a single lane at least one time per study In the figure below CS signifies a Sub CodeSet CSI CS2 A single reference sample red tube marked one is assayed in each Sub CodeSet once per study All other tubes contain unique samples i IMPORTANT Be sure to only mix only unique sub CodeSets in a single tube Use any given sub CodeSet only one time in a single lane of the cartridge The final hybridization reaction will contain the following components 10L Reporter CodeSet 10uL hybridization buffer a total vol
10. exists BOLD When appearing in text or in a procedure the bold text serves to highlight a specific button key stroke or menu option available Bold text may appear elsewhere to highlight important text or terms Green text is used to help the reader identify active hyperlinks ITALICS Used to emphasize an important word or expression within the text Formatting of a book title journal or other documentation Used to indicate the special or unusual meaning of a word or phrase Procedures Numbered procedures appear frequently providing step by step instruction for accomplishing a task Typically a numbered step provides direction for a specific action and may be followed by the expected response Additional information may be presented in the form of a specific note type bullets screen capture or other image important to facilitate clarity and understanding For example In the next screen the active data entry field is indicated by a green box around it Simply move from one field to the next simply press the desired field on the touchscreen with your finger 1 To add an email address press ADD gt gt gt The email address keyboard screen appears 2 Enter a valid email address and press ENTER The email address gets saved Contact Information NanoString Technologies Inc 530 Fairview Ave N Suite 2000 Seattle Washington 98109 USA Tel 206 378 6266 888 358 NANO 6266 Fax 206 378 6288 Email su
11. hermocycler and combine full hybridization volumes of strip tubes CS2 CS3 and CS4 into CS1 strip tube 1 as shown in Figure 2 4 maintaining tube orientation The resulting final volume is 120uL per well 13 Once removed from the thermocycler recap and briefly spin tubes before proceeding immediately to post hybridization processing with the nCounter Prep Station Do not store hybridizations at 4 C FIGURE 2 4 Post hybridization mixing step Post hybridization add full hybridization reaction volume from CS2 CS3 and CS4 into the CS1 tube maintaining tube orientation The resulting final volume is 120uL per well In the figure above hybridization volumes from CS1 CS2 and CS3 are combined into the CS1 strip tube The CS1 strip tube is then recapped briefly spun and loaded into the Prep Station Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation 17 USER MANUAL nCounter Gene Expression Assay NanoString Technologies Inc 530 Fairview Ave N Suite 2000 Seattle Washington 98109 2013 NanoString Technologies Inc All rights reserved FOR RESEARCH USE ONLY Not for use in diagnostic procedures CONTACT US info nanostring com Tel 888 358 6266 Fax 206 378 6288 www nanostring com nanoString SALES CONTACTS United States us sales nanostring com Europe europe sales nanostring com Other Regions info nanostring com 20131213 MAN C0003 03
12. ifuge to go to maximum speed and you may spin your CodeSet out of solution i IMPORTANT To facilitate downstream analysis in the nSolver Analysis System the same sample Calibration sample must be run across both CodeSets in the same lane at least once per study see Figure 2 3 This means that you must hybridize the same Calibration sample in both sub CodeSets one time For more information on the Calibration sample see the nCounter Expression Data Analysis Guidelines FIGURE 2 3 Hybridization setup To facilitate downstream analysis in the nSolver Analysis System a Calibration sample must be assayed across all four Sub CodeSets in a single lane at least one time per study In the figure below CS signifies a Sub CodeSet CSI CS4 A single Calibration sample red tube marked one is assayed in each Sub CodeSet at least once per study All other tubes contain unique samples i IMPORTANT Be sure to only mix only unique sub CodeSets in a single tube Use any given sub CodeSet only one time in a single lane of the cartridge The final hybridization reaction will contain the following components 1OyL Reporter CodeSet 10uL hybridization buffer a total volume of SuL of sample RNA 150ng and 5uL Capture ProbeSet The order of addition of components is important please follow the protocol exactly 1 If following the Total RNA Standard Protocol go to Step 3 2 If following the Cell Lysate Protocol Lyse Cells according to
13. ing to Qiagen recommendations see Qiagen RNeasy Mini Handbook supplied with product numbers 74104 and 74106 with the following modifications a Cells should be lysed at concentration between 2 500 and 10 000 cells uL of RLT buffer The nCounter cell lysate hybridization procedure has been optimized for 10 000 mammalian cells reaction or the equivalent of approximately 100ng of total RNA b Cell lysates should be aliquoted and stored at 80 C Avoid freeze thaw cycles 3 Remove aliquots of both Reporter CodeSet and Capture ProbeSet reagent from the freezer and thaw on ice Invert several times to mix well and spin down reagent i IMPORTANT After it has thawed inspect the tube of Reporter CodeSet to make sure no colored precipitate is present If you see a colored precipitate heat the entire tube to 75 C for 10 minutes and cool at room temperature before using 4 Create a master mix by adding 130uL of hybridization buffer to the tube of Reporter Probes Do not remove the Reporter Probes from tube RNAse free water may also be added to this mix if the volume of the individual RNA samples is less than 5uL and is constant Add enough water for 13 assays to allow one assay s worth of dead volume Do not add the Capture ProbeSet to the master mix Invert to mix and spin down master mix 10 NanoString Technologies NanoString Technologies USER MANUAL 5 Label a provided 12 tube strip and cut it in half so it will fit in a picof
14. ion miRNA Expression Copy Number Variation a nCounter Gene Expression Assay Setting Up a Single nCounter Assay During setup of your assay do not vortex or pipet vigorously to mix as it may shear the Reporter Probes Mixing should be done by flicking or inverting the tubes Also if you use a microfuge to spin down tubes do not spin any faster than 1 000 rpm for more than 30 seconds and do not pulse it to spin because that will cause the centrifuge to go to maximum speed and you may spin your CodeSet out of solution The final hybridization reaction will contain the following components 10uL Reporter CodeSet 10uL hybridization buffer a total volume of Sul of sample RNA 100ng and 5uL Capture ProbeSet If you are making a master mix of any components DO NOT include the Capture ProbeSet It is important that the Capture ProbeSet be added individually to each assay immediately before the reaction is transferred to 65 C 1 If following the Total RNA Standard Protocol go to Step 3 2 If following the Cell Lysate Protocol Lyse Cells according to Qiagen recommendations see Qiagen RNeasy Mini Handbook supplied with product numbers 74104 and 74106 with the following modifications a Cells should be lysed at concentration between 2 500 and 10 000 cells uL of RLT buffer The nCounter cell lysate hybridization procedure has been optimized for 10 000 mammalian cells reaction or the equivalent of approximately 100ng of total RNA
15. med and excess Capture Probes are washed away Finally the purified Target Probe complexes are eluted off and are immobilized in the cartridge for data collection Data Collection is carried out in the nCounter Digital Analyzer At the highest standard data resolution 555 1155 fields of view FOV are collected per flow cell using a microscope objective and a CCD camera yielding data of hundreds of thousands of target molecule counts Digital images are processed on the nCounter Digital Analyzer and the barcode counts are tabulated in a comma separated value CSV format The nCounter Analysis System was created by NanoString Technologies The nCounter system is an easy to use integrated system that includes a Prep Station robot and a Digital Analyzer analyzer The Prep Station and the analyzer together make lab work and sample analysis a simpler process by limiting the variables in experiments for lab technicians The end result is a very precise and accurate measurement enabling you to gather data on your targets of interest rapidly with minimal intervention nCounter Expression Assay Overview The nCounter Expression Assay is run on the nCounter System The system is comprised of two instruments the nCounter Prep Station used for post hybridization processing and the Digital Analyzer used for data collection Follow the instructions on the touchscreen to guide you step by step through setting up runs on the Prep Station and Digital Analyzer
16. nCounter Gene Expression Assay User Manual Total RNA and Cell Lysate Protocols NanoString Technologies Inc 530 Fairview Ave N Suite 2000 Seattle Washington 98109 www nanostring com Tel 206 378 6266 888 358 6266 E mail info nanostring com Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation MAN C0003 03 nCounter Gene Expression Assay FOR RESEARCH USE ONLY Not for use in diagnostic procedures Intellectual Property Rights This nCounter Analysis System manual and its contents are the property of NanoString Technologies Inc NanoString and is intended solely for the use of NanoString customers for the purpose of operating the nCounter Analysis System The nCounter Analysis System including both its software and hardware components and this User Guide and any other documentation provided to you by NanoString in connection therewith are subject to patents copyright trade secret rights and other intellectual property rights owned by or licensed to NanoString No part of the software or hardware may be reproduced transmitted transcribed stored in a retrieval system or translated into other languages without the prior written consent of NanoString Limited License Subject to the terms and conditions of the nCounter Analysis System contained in the product quotation NanoString grants you limited non exclusive non transferable non
17. obe pair The probe pair consists of a Reporter Probe which carries the signal on its 5 end and a Capture Probe which carries a biotin on the 3 end The color codes carry six positions and each position can be one of four colors thus allowing for a large diversity of tags that can be mixed together in a single well for direct hybridization to target and yet still be individually resolved and identified during data collection FIGURE 1 1 Capture and Reporter Probes left and Probe pair bound to an mRNA right CAPTURE PROBE REPORTER PROBE TARGET PROBE COMPLEX TARGET 6 NanoString Technologies NanoString Technologies USER MANUAL Probe pairs are placed into a reaction in massive excess to target RNA or DNA species to ensure that each target finds a probe pair After hybridization excess probes are washed away using a two step magnetic bead based purification on the nCounter Prep Station Magnetic beads derivatized with short nucleic acid sequences that are complementary to the Capture Probe and the Reporter Probes are used sequentially First the hybridization mixture is allowed to bind to the magnetic beads by the Capture Probe Wash steps are performed and excess Reporter Probes and non target cellular transcripts are removed during wash steps After washing the Capture Probes and Target Probe complexes are eluted off of the beads and are hybridized to magnetic beads complementary to the Reporter Probe Wash steps are perfor
18. ogram the thermocycler using 30yL volume calculated temperature heated lid and forever time setting Do not set the thermocycler to ramp down to 4 C at the end of the run If a thermocycler is not available a 65 C hybridization oven may be used The use of a thermocycler is recommended if possible Due to less stringent temperature control assay results may be more variable in a hybridization oven To use a hybridization oven place a large beaker full of water in the oven to ensure a humid environment Do not place the samples in the water beaker evaporative loss causes the water temperature to be below the air temperature in the oven Place the samples in a dry rack in the middle of the oven shelf or tape the strip tubes to a rotator in the center of the oven Make sure that the strip tubes and or rack do not touch the sides or bottom of the oven Failure to follow these instructions may result in uneven hybridization temperatures which can compromise the results 10 Add 5uL of Capture ProbeSet to each tube immediately before placing at 65 C Cap tubes and mix the reagents by inverting the strip tubes several times and flicking with your finger to ensure complete mixing Briefly spin down and immediately place the strip tube in the 65 C thermocycler Minimizing the time between the addition of the Capture ProbeSet and the placement of the reaction at 65 C will increase the sensitivity of your assay 11 Incubate hybridization assays fo
19. pport nanostring com nanoString NanoString Technologies USER MANUAL This page intentionally left blank Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation 5 nCounter Gene Expression Assay Introduction Introduction The nCounter Gene Expression Assay is designed to provide an sensitive reproducible and highly multiplexed method for detecting gene expression across all levels of biological expression This assay provides a method for detecting mRNAs with molecular barcodes called nCounter Reporter Probes without the use of reverse transcription or amplification This manual describes in detail the methods for setting up nCounter hybridization assays Please see the nCounter Prep Station User Manual nCounter Digital Analyzer User Manual and nCounter Data Analysis Guidelines for instructions on post hybridization processing and data analysis This manual covers the nCounter Gene Expression Assay and Plex Gene Expression Assay protocols and provides instruction for both the Total RNA Standard Protocol and the Cell Lysate Protocol The Total RNA Standard Protocol can also be used with total RNA isolated from blood and Formalin Fixed Paraffin Embedded FFPE samples NanoString Technology Principles and Procedures NanoString s technology is based on digital detection and direct molecular barcoding of target molecules through the use of a color coded pr
20. r at least 12 hours Hybridizations should be left at 65 C until ready for processing Maximum hybridization time should not exceed 30 hours 12 Remove tubes from the thermocycler and combine full hybridization volumes of strip tubes 1 and 2 into strip tube 1 as shown in Figure 2 2 maintaining tube orientation The resulting final volume is 6OuL per well 13 Once removed from the thermocycler recap tubes briefly spin and proceed immediately to post hybridization processing with the nCounter Prep Station Do not store hybridizations at 4 C FIGURE 2 2 Post hybridization mixing step rye i a a a a a eed LLL yyw WwW WwwwyY iY Post hybridization add full hybridization reaction volume from CS2 into the CS1 tube maintaining tube orientation The resulting final volume is 60u1L per well In the figure above hybridization volumes from CS2 are combined into the CS1 strip tube The CS1 strip tube is then recapped and briefly spun before loading into the Prep Station nanoString NanoString Technologies USER MANUAL Setting Up 48 Plex Assays 4PLEX A GENERAL PROBE HANDLING WARNING During setup of your assay do not vortex or pipet vigorously to mix as it may shear the Reporter Probes Mixing should be done by flicking or inverting the tubes Also if you use a microfuge to spin down tubes do not spin any faster than 1 000 rpm for more than 30 seconds and do not pulse it to spin because that will cause the centr
21. r to ramp down to 4 C at the end of the run Alternatively a 65 C hybridization oven may be used when a large beaker full of water is placed in the oven to ensure a humid environment 10 Add 5uL of Capture ProbeSet to each tube immediately before placing at 65 C Cap tubes and mix the reagents by inverting the strip tubes several times and flicking with your finger to ensure complete mixing Briefly spin down and immediately place the strip tube in the 65 C thermocycler Minimizing the time between the addition of the Capture ProbeSet and the placement of the reaction at 65 C will increase the sensitivity of your assay 11 Store remaining Capture ProbeSet at 4 C for up to 1 month 12 Incubate hybridization assays for at least 12 hours Hybridizations should be left at 65 C until ready for processing Maximum hybridization time should not exceed 30 hours 13 Once removed from the thermocycler proceed immediately to post hybridization processing with the nCounter Prep Station Do not store hybridizations at 4 C nanoString 12 3 i p s NanoString Technologies USER MANUAL This section outlines the nCounter Plex Expression Assay protocol and provides instruction for both the Total RNA Standard Protocol and the Cell Lysate Protocol Instruction for setting up 24 and 48 Plex Assays 1 cartridge is also provided Setting Up 24 Plex Assays 2PLEX A GENERAL PROBE HANDLING WARNING During setup of your assay do not vort
22. rt to mix and spin down master mix 5 Label four provided 12 tube strip tubes and cut each in half so it will fit in a picofuge 6 Add 20uL of mastermix to each of the tubes if you added water to the master mix adjust volumes It is advisable to use a fresh tip for each pipetting step to accurately pipet the correct volume The CodeSet has components that can start to wick up into the tip and not dispense the correct amount if you use the same tip to dispense master mix into all of the hybridization tubes 7 Add sample according to your protocol type as follows a If following the Total RNA Standard Protocol Add total RNA sample maximum volume Sy for a total of 15Ong to each tube Go to Step 8 b If following the Cell Lysate Protocol Add cell lysate sample maximum volume 4yL for a total of approximately 15 000 cells per hybridization assay Using less than 15 000 cells reaction will result in fewer counts gene c If using attenuation mix es add 1uL of each mix Note this reagent can also be added to the master mix if all reactions are to be attenuated 8 If necessary add RNAse free water to each tube to bring the volume of each assay to 25uL 9 Pre heat thermocycler to 65 C Program the thermocycler using 30yL volume calculated temperature heated lid and forever time setting Do not set the thermocycler to ramp down to 4 C at the end of the run If a thermocycler is not available a 65 C hybridization oven may b
23. tion oiai NanoString Technology Principles and Procedures nCounter Gene Expression Assay Overview Materials Required Materials required for Total RNA Standard Protocol Materials required for Cell Lysate Protocol Instruments required for Total RNA Standard Protocol and Cell Lysate Protocol Contact Information Chapter 2 nCounter Gene Expression Protocols Setting Up 12 nCounter Assays Setting Up a Single nCounter Assay Setting Up 24 Plex Assays Setting Up 48 Plex Assays Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation 3 nCounter Gene Expression Assay PREFACE Conventions Used The following conventions are used throughout this manual and are described below for your reference Note Types Special font formatting is used in this manual Such formatting conventions are used in specific instances as described below TIP Information contained in a Tip may offer helpful suggestions alternative procedures methods and or shortcuts NOTE This note type emphasizes general information IMPORTANT i This note type presents essential content indicating that the potential exists for assay failure diminished data quality and or a loss of data if the information presented is ignored WARNING This note type indicates that a potential hazard to your personal safety or the potential for equipment damage
24. tring Technologies Inc 530 Fairview Ave N Suite 2000 Seattle Washington 98109 Tel 888 358 6266 Fax 206 378 6288 E mail support nanostring com Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation nCounter Gene Expression Assay Gene Expression Protocols This chapter outlines the nCounter Gene Expression Assay protocol and provides instruction for both the Total RNA Standard Protocol and the Cell Lysate Protocol Instruction for setting up 12 nCounter Assays or a single nCounter Assay are provided Setting Up 12 nCounter Assays A GENERAL PROBE HANDLING WARNING During setup of your assay do not vortex or pipet vigorously to mix as it may shear the Reporter Probes Mixing should be done by flicking or inverting the tubes Also if you use a microfuge to spin down tubes do not spin any faster than 1 000 rpm for more than 30 seconds and do not pulse it to spin because that will cause the centrifuge to go to maximum speed and you may spin your CodeSet out of solution The final hybridization reaction will contain the following components 10uL Reporter CodeSet 10uL hybridization buffer a total volume of SuL of sample RNA 100ng and 5uL Capture ProbeSet The order of addition of components is important please follow the protocol exactly 1 If following the Total RNA Standard Protocol go to Step 3 2 If following the Cell Lysate Protocol Lyse Cells accord
25. uge 6 Add 20uL of mastermix to each of the 12 tubes if you added water to the master mix adjust volumes It is advisable to use a fresh tip for each pipetting step to accurately pipet the correct volume The CodeSet has components that can start to wick up into the tip and not dispense the correct amount if you use the same tip to dispense master mix into all of the hybridization tubes 7 Add sample according to your protocol type as follows a If following the Total RNA Standard Protocol Add total RNA sample maximum volume 5uL for a total of 100ng to each tube Go to Step 8 b If following the Cell Lysate Protocol Add cell lysate sample maximum volume 4yL for a total of approximately 10 000 cells per hybridization assay Using less than 10 000 cells reaction will result in fewer counts gene c If using attenuation mix es add 1uL of each mix Note this reagent can also be added to the master mix if all reactions are to be attenuated 8 If necessary add RNAse free water to each tube to bring the volume of each assay to 25uL 9 Pre heat thermocycler to 65 C Program the thermocycler using 30yL volume calculated temperature heated lid and forever time setting Do not set the thermocycler to ramp down to 4 C at the end of the run If a thermocycler is not available a 65 C hybridization oven may be used The use of a thermocycler is recommended if possible Due to less stringent temperature control assay results may
26. ume of 5uL of sample RNA 100ng and 5uL Capture ProbeSet The order of addition of components is important please follow the protocol exactly 1 If following the Total RNA Standard Protocol go to Step 3 2 If following the Cell Lysate Protocol Lyse Cells according to Qiagen recommendations see Qiagen RNeasy Mini Handbook supplied with product numbers 74104 and 74106 with the following modifications a Cells should be lysed at concentration between 2 500 and 10 000 cells uL of RLT buffer or similar The nCounter cell lysate hybridization procedure has been optimized for 10 000 mammalian cells reaction or the equivalent of approximately 100ng of total RNA b Cell lysates should be aliquoted and stored at 80 C Avoid freeze thaw cycles 3 Remove aliquots of both Reporter CodeSet and Capture ProbeSet reagent from the freezer and thaw on ice Invert several times to mix well and spin down reagent i IMPORTANT After it has thawed inspect the tube of Reporter CodeSet to make sure no colored precipitate is present If you see a colored precipitate heat the entire tube to 75 C for 10 minutes and cool at room temperature before using Molecules That Count Translational Research Gene Expression miRNA Expression Copy Number Variation 13 nCounter Gene Expression Assay 4 Create a master mix by adding 130uL of hybridization buffer to the tube of Reporter Probes Do not remove the Reporter Probes from tube RNAse free
27. water may also be added to this mix if the volume of the individual RNA samples is less than 5uL and is constant Add enough water for 13 assays to allow one assay s worth of dead volume Do not add the Capture ProbeSet to the master mix Invert to mix and spin down master mix 5 Label two provided 12 tube strip tubes and cut each in half so it will fit in a picofuge 6 Add 20uL of mastermix to each of the 24 tubes if you added water to the master mix adjust volumes It is advisable to use a fresh tip for each pipetting step to accurately pipet the correct volume The CodeSet has components that can start to wick up into the tip and not dispense the correct amount if you use the same tip to dispense master mix into all of the hybridization tubes 7 Add sample according to your protocol type as follows a If following the Total RNA Standard Protocol Add total RNA sample maximum volume 5 uL for a total of 100ng to each tube Go to Step 8 b If following the Cell Lysate Protocol Add cell lysate sample maximum volume 4L for a total of approximately 10 000 cells per hybridization assay Using less than 10 000 cells reaction will result in fewer counts gene c If using attenuation mix es add 1uL of each mix Note this reagent can also be added to the master mix if all reactions are to be attenuated 8 If necessary add RNAse free water to each tube to bring the volume of each assay to 25uL 9 Pre heat thermocycler to 65 C Pr
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