NanoString®: MANUAL
Contents
1. 5 Denature samples from the miRNA sample prep protocol at 85 C for 5 minutes and quick cool on ice Add a 5 uL aliquot from the miRNA Sample Preparation Protocol to each tube Molecules That Count Translational Research e Gene Expression e miRNA Expression es Copy Number Variation e Single Cell 13 USER MANUAL nCounter miRNA Expression Assay 6 Pre heat thermocycler to 65 C Program the thermocycler using 30 uL 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 NOTE 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 7 Add 5 uL of Capture ProbeSet to each tube immediately before placing a2. GIE SI USER MANUAL miIRNA Sample Preparation Protocol The nCounter miRNA Expression Assay requires purified total RNA as input material NanoString recommends the use of 100 ng total RNA as this quantity of input material generates robust signal for most tissue cell isolates Purified RNA quality is also important as residual organics phenol etc can impact assay performance NanoString recommends a minimum 280 260 ratio of 1 9 and a minimum 260 230 ratio of 1 8 All experiments should be designed in sets of twelve assays The protocol below is for one set of 12 assays All reagents are supplied in 12 reaction aliquots 1 Normalize RNA samples to 33 ng uL using DEPC or RNAse free HO 2 Prepare a 1 500 dilution of the miRNA Assay Controls To do this we recommend adding 499 uL DEPC H O to 1 uL of the miRNA Assay Controls in a sterile microcetrifuge tube Mix by vortexing and briefly spin down Store on ice 3 Prepare an annealing master mix by combining 13 uL of Annealing Buffer 26 uL of nCounter miRNA Tag Reagent and 6 5 uL of the 1 500 miRNA Assay Controls dilution prepared in Step 2 Mix well by pipetting up and down 4 Aliquot 3 5 uL of the annealing master mix into each tube of a 12 x 0 2 mL strip tube 5 Add 3 uL 100 ng of RNA sample to each tube Cap tubes and flick tubes gently to mix Spin down 6 Place strip in thermocycler and initiate Annealing Protocol 7 Combine 19 5 uL PEG and 13 uL Ligation Buffer to prepa
3. Preparation Protocol nanostring 8 T E C H N LO GIE SI NanoString Technologies USER MANUAL Sample Guidelines Recommendations The nCounter miRNA Expression Assay requires purified total RNA as input material NanoString recommends the use of approximately 100 ng of total RNA as this quantity of inout material generates robust signal for most tissue and cell isolates Total RNA purified from any cell or tissue type may be used in the assay including formalin fixed paraffin embedded FFPE material Unpurified lysates may not be used with the nCounter miRNA Expression assay as the denaturants in the homogenization buffer will inhibit the sample preparation reaction The quality of the purified RNA is critically important for the nCounter miRNA assay as residual contaminants left over from lysis and RNA extraction can impact assay performance by inhibiting the enzymatic ligation and purification steps Typical lysis or extraction contaminants that can inhibit the assay Include e Guanidinium Isothiocyanate lysis buffer e Phenol organic extraction e Guanidinium HCI initial wash buffer e Ethanol secondary wash buffer Purified RNA quality can be evaluated via a spectrophotometer by measuring absorbance at 230 nm A_ 260 nm A and 280 nm CA _ 230 260 280 The Asd A ratio can help identify contamination with proteins whereas the A AN ratio can help identify contamination with organic 230 compounds such
4. USE ONLY Not for use in diagnostic procedures MAN C0009 05 EE E EK ES EE NEESS penaing patent applications owne
5. e Gene Expression e miRNA Expression e Copy Number Variation e Single Cell 3 USER MANUAL nCounter miRNA Expression Assay PREFACE Purpose This user manual details specific information pertaining to safe and proper handling operation and maintenance of the nCounter Digital Analyzer 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 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 A This note type indicates that a potential hazard to your personal safety or the potential for equipment damage 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 e 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 docume
6. Kit allows the user to monitor the ligation efficiency and specificity through each step of the reaction The total hands on time for the sample preparation reaction is approximately 30 minutes with an elapsed time of approximately 2 hours FIGURE 1 1 miRNAs are specifically ligated to unique tags for downstream detection Mature miRNA PA Ligation Site miRtag Sequence 6 NanoString Technologies Inc NanoString Technologies USER MANUAL CodeSet Hybridization and Downstream Processing Overview NanoString technology is based on the direct molecular barcoding and digital detection of target molecules through the use of a color coded orobe 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 its A end The complexity of the color codes comprised of four colors in six positions allows a large diversity of targets present in the same sample to be individually resolved and identified during data collection During the overnight hybridization reaction probe pairs are present in large excess to target RNAs to ensure that each target finds a probe pair FIGURE 1 2 Capture and Reporter Probes top and Probe pair bound to an RNA target bottom Capture Probe Reporter Probe Target Probe Complex The nCounter miRNA expression assay is run on the nCounter Analysis system The system is comprised of two instruments the Prep Station use
7. MANUAL EE 4 feele 4 ei E ME 4 gt akk a a gt errkrRTRiir rooreE e eKrrxrrva _K lJJJnmnnn 4 gt gt gt 555555 55 D5n2n DnD D 5D DADA A o m mm 4 No gt o oD 7Don DDD2 nD D JJJrrrrmrmdmararr A 4 Bau E een O rddidi 4 Chapter 1 Introduction EEN 6 10 The nCounter MIRNA Expression Assay l lkae EEEE KEEKEEKE EEEE EEEE EEEE EEEEEEE kaka kak kak kak A 6 MIRNA Sample Preparation OvervieW 0 ccccccsscccsseceesecerseceereeseeecesecnseeseeeeneeesnreeeniueettevenessnesentesenteses 6 CodeSet Hybridization and Downstream Processing OVerV W cece cceecessceescessesesssessesenneernennen 7 Materials Requi red DE 8 Sample Guidelines Recommendations eee keyey eyek nane 9 Understanding and Troubleshooting RNA Sample Contamination nnn 9 Thermocycler Requirement oo cccccccccccccccccccescecceccscecceccsececceceesercevsesersereveesarsetsesaesarcessesarsescatusersersavarsareatersanees 10 Thermocycler PO EO CONS passes cases seeps eccee tees s aceon cu tc secs asainpeseediegenceens emeotssoacenestees 10 Chapter 2 miRNA Sample Preparation Protocol 11 12 Chapter 3 MIRNA Hybridization Protocol ooo ccccsecsesesererestreserenrersetrsistrersisreresrersnrerseres 13 14 Setting Up nCounter Hybridization Assays cc ccc ceecceseeeseseseeceeeceuessesseecauessueseseseenesnesneses 13 Molecules That Count Translational Research
8. T a C nanostr i H N O L O nCounter nCounter miRNA Expression Assay User Manual Sample Preparation and Hybridization Protocols NanoString Technologies Inc 530 Fairview Ave N Suite 2000 Seattle Washington 98109 www nanostring com Tel 206 5 8 6266 888 558 6266 E mail info nanostring com Molecules That Count Translational Research e Gene Expression miRNA Expression e Copy Number Variation e Single Cell MAN COQ009 05 USER MANUAL nCounter miRNA 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 conta
9. as phenol and guanidinium salts NanoString recommends a 260 280 ratio of 1 9 or greater and a 260 230 ratio of 1 8 or greater for optimal results Understanding and Troubleshooting RNA Sample Contamination Significant absorbance at 280 nm can indicate contamination with protein Such contamination may lead to an overestimation of the RNA concentration resulting in a lower than anticipated signal in the assay Significant absorbance at 230 nm is indicative of contamination with phenol or guanidinium a pure RNA sample should have a A A ratio above 2 0 Extra washes with a secondary wash buffer or ethanol can help to minimize carry though It is also important that residual secondary wash buffer be removed prior to elution resuspension NOTE At very low RNA concentrations under 10 ng uL the A A ratio may be unreliable as an indicator of contamination due to limited nucleic acid absorbance at 260 nm NanoString recommends preparing samples with a concentration of gt 33 no HL allowing 100 ng of total RNA to be added to the sample preparation reaction in the available 3 uL volume Some RNA extraction protocols suggest that better yield can be achieved by re eluting the column with the initial eluate NanoString does not recommend this as the extra elution can generate significant carry through of guanidinium and organic contamination If re elution must be performed it should be preceded by at least 2 additional column washes with t
10. d for detecting miRNAs in total RNA across all biological levels of expression The assay provides a method for detecting miRNAs without the use of reverse transcription or amplification using molecular barcodes called nCounter Reporter Probes The assay can be run on total RNA isolated from any source including formalin fixed paraffin embedded FFPE samples This manual describes in detail the methods for both the miRNA Sample Preparation and the miRNA CodeSet Hybridization For instructions on post hybridization processing and data analysis please see the nCounter Prep Station User Manual nCounter Digital Analyzer User Manual and nCounter Data Analysis Guidelines for miRNA The nCounter miRNA Expression Assay miRNA Sample Preparation Overview The nCounter miRNA Sample Preparation Kit provides reagents for ligating unique oligonuceotide tags onto miRNAs allowing these short RNAs to be detected with great specificity and sensitivity in the standard nCounter gene expression assay The miRNA tag ligation reaction can be performed in a background of total RNA Sample preparation involves a multiplexed annealing of the specific tags to their target miRNA a ligation reaction and an enzymatic purification to remove the unligated tags Sequence specificity between each miRNA and its appropriate tag is ensured by careful stepwise control of annealing and ligation temperatures Control RNA included in the nCounter Human miRNA Sample Preparation
11. d for post hybridization processing and the Digital Analyzer used for data collection 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 containing target probe complexes is allowed to bind to magnetic beads complementary to sequences on the Capture Probe Wash steps are performed to remove excess Reporter Probes and non target cellular transcripts After washing the Capture Probes and target probe complexes are eluted off the beads and are hybridized to magnetic beads complementary to sequences on the Reporter Probe An additional wash is performed to remove excess Capture Probes Finally the purified target probe complexes are eluted off the beads and immobilized on the cartridge for data collection Data Collection is carried out in the nCounter Digital Analyzer Digital images are processed and the barcode counts are tabulated in a comma separated value CSV format FIGURE 1 2 Suggested workflow for the nCounter miRNA Expression Assay Manual Processing Hands on Time miRNA Sample Preparation 30 minutes Day 1 R j Set Up Hybridization 5 minutes Set Up Prep Station Run 5 minutes Day 2 SE Set Up Data Collection 5 minutes Molecules That Count Translational Research e Ge
12. he secondary ethanol based wash buffer for a total of 4 secondary washes Ethanol is not evident spectrophotometrically but can be eliminated by a one minute post wash centrifugation in a clean collection tube as is suggested in most kit protocols Additionally air drying the filter for five minutes can help if ethanol contamination persists Molecules That Count Translational Research e Gene Expression e miRNA Expression e Copy Number Variation e Single Cell 9 USER MANUAL nCounter miRNA Expression Assay Thermocycler Requirement The thermocycler used for the miRNA Sample Preparation Protocol must have a heated lid For best results NanoString recommends calibrating the thermocycler before using this assay Thermocycler Protocols The nCounter miRNA Preparation protocol requires careful temperature control of all reaction steps A thermocycler with a heated lid is critical for this procedure Before beginning program the following thermocycler protocols TABLE 1 3 Annealing Protocol Temperature Time 94 C 1 min 65 C 2 min 45 C 10 min 48 C hold Total Time 13 min TABLE 1 4 Ligation Protocol Temperature Time 48 C 3 min 47 C 3 min 46 C 3 min 45 C 5 min 65 C 10 min 4 C hold Total Time 24 min TABLE 1 5 Purification Protocol Temperature Time 37 C 1 hour 70 C 10 min 4 C hold Total Time 1 hour 10 min nanostring 10 T E C H N LO
13. ined in the product quotation NanoString grants you a limited non exclusive non transferable non 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 2 T E C H N LO GIE SI NanoString Technologies USER
14. k close thermocycler and initiate Ligation Protocol 12 After completion of Ligation Protocol add 1 uL Ligation Clean Up Enzyme to each reaction The tubes can be removed from the heat block for this step Flick tubes gently to mix Spin down 13 Return tubes to thermocycler and initiate Purification Protocol 14 After completion of Purification Protocol add 40 uL DEPC or RNAse free H O to each sample Mix well and spin down If necessary at this stage purified sample preparation reactions may be stored at 20 C for up to several weeks Be sure to denature Step 5 of Chapter 3 miRNA Hybridization Protocol before adding prepared sample in the hybridization protocol 15 Proceed immediately with the miRNA CodeSet Hybridization Protocol which is described in Chapter 3 of this manual nanostring 12 T E C H N LO GIE SI USER MANUAL miIRNA Hybridization Protocol This chapter outlines the nCounter miRNA Hybridization Protocol and provides instructions for setting up 12 nCounter Assays Setting Up nCounter Hybridization Assays A GENERAL PROBE HANDLING WARNING During the setup of your assay do not vortex or pipet reactions vigorously to mix as it may shear the Reporter Probes Mixing should be done by flicking or inverting the tubes Do not spin tubes any faster than 1 000 rom for more than 30 seconds do not pulse microfuge to spin as that will cause the centrifuge to go to maximum speed and you may spin your C
15. ne Expression e miRNA Expression e Copy Number Variation e Single Cell 7 USER MANUAL nCounter miRNA Expression Assay Materials Required The following tables list the recommended materials and instrumentation required to run the nCounter miRNA Expression Assay TABLE 1 1 Materials and Reagents Required for miRNA Sample Preparation and miRNA Expression Assay Material Manufacturer nCounter miRNA Expression Assay Kit NanoString Technologies Total RNA Extraction Kit QIAGEN or equivalent total RNA purification kit Disposable gloves various DEPC treated or RNA se free water 100ng total RNA per sample normalized to 33ng uL TABLE 1 2 Instruments Required for miRNA Sample Preparation and miRNA Gene Expression Assay Material Manufacturer Spectrophotometer NanoDrop Technologies or equivalent Bioanalyzer 2100 Agilent Pipette for 0 5 10 uL Rainin or equivalent Pipette for 2 0 20 uL Rainin or equivalent Pipette for 20 200 uL Rainin or equivalent Picofuge with strip tube adaptor Stratagene or equivalent DNA Engine Thermocycler or hybridization oven MJ Research Bio Rad or equivalent nCounter Prep Station NanoString Technologies nCounter Digital Analyzer NanoString Technologies USB Drive NanoString Technologies Hybridization oven can be used for the miRNA Hybridization Protocol only A thermocycler with a heated lid is required for the miRNA Sample
16. ntation e 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 206 578 0206 888 558 NANO 6266 Fax 206 378 6288 Email support nanostring com nanostring 4 T E C H N LO GIE SI NanoString Technologies USER MANUAL Molecules That Count Translational Research e Gene Expression e miRNA Expression e Copy Number Variation e Single Cell 5 nCounter miRNA Expression Assay Introduction Introduction The nCounter miRNA Expression Assay is designed to provide an ultra sensitive reproducible and highly multiplexed metho
17. odeSet out of solution The final hybridization reaction will contain the following components 10 uL Reporter CodeSet 10 uL hybridization buffer a 5 uL aliquot from the miRNA Sample Preparation Protocol and 5 uL Capture ProbeSet The order of addition of components is important please follow the protocol exactly 1 Remove aliquots of both the Reporter CodeSet and Capture ProbeSet reagent from the freezer and thaw on ice Invert several times to mix well and briefly spin down reagent at lt 1000 rom see handling considerations above 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 2 Create a master mix containing 130 uL of the Reporter CodeSet and 130 uL of hybridization buffer by adding the hybridization buffer to the tube containing the Reporter CodeSet Do not add the Capture ProbeSet to the master mix Invert to mix and spin down master mix Label a provided 12 tube strip and cut it in half so it will fit in a picofuge 4 Add 20 uL of master mix to each of the 12 tubes NOTE It is advisable to use a fresh tip for each pipetting step to 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
18. re a ligation master mix NOTE PEG is viscous and should be pipetted slowly to ensure accurate transfer of volume into the mix Mix well by pipetting up and down 8 Following completion of the Annealing Protocol when the thermocycler has reached 48 C add 2 5 uL of the ligation master mix to each tube Do not turn off the thermocycler you will need the block to be at 48 C in Step 9 and Step 10 Flick tubes gently to mix and spin down 9 Return tubes to 48 C thermocycler close lid and incubate at 48 C for 5 min A WARNING For Step 10 DO NOT REMOVE TUBES FROM THE THERMOCYCLER Maintaining the temperature of the tubes at 48 C is critical for optimal assay performance Tubes should never be removed from heat block during this step 10 Open thermocycler carefully remove caps from tubes leaving strip in place in the heat block and add 1 0 uL of Ligase directly to each tube while incubating at 48 C Check the pipette tip to make certain all of the ligase was added to the reaction There is no need to mix Molecules That Count Translational Research e Gene Expression e miRNA Expression es Copy Number Variation e Single Cell 11 USER MANUAL nCounter miRNA Expression Assay NOTE To keep track of Ligase addition to sequential samples it can be helpful to line up 12 tips in front of thermocycler discarding each tip after use 11 Immediately after addition of Ligase to the final tube recap tubes leaving tubes in heat bloc
19. t 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 at lt 1000 rom 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 8 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 9 Once removed from the thermocycler proceed immediately to post hybridization processing with the nCounter Prep Station as described in the nCounter Prep Station User Manual Do not store hybridizations at 4 C NanoString Technologies Inc CONTACT US SALES CONTACTS 530 Fairview Ave N info nanostring com United States us sales nanostring com Suite 2000 Tel 888 358 6266 Europe europe sales nanostring com Seattle Washington 98109 Fax 206 378 6288 Japan japan sales nanostring com www nanostring com Other Regions info nanostring com 2013 NanoString Techn SrA PAAleraAran tranamar are registered trade lal ologies Inc All righ or trademarks of NanoString Te J PE N ven NO VI cd E nT K a e CT ae se k n ll Sn Tun la yev wila H D icensed to NanoString from Life Technologies d by NanoString or J FOR RESEARCH
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