ncounter Elements TagSets General Purpose Reagents PACKAGE INSERT Molecules That Count NanoString Technologies, Inc.

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1 ncounter Elements TagSets General Purpose Reagents PACKAGE INSERT NanoString Technologies, Inc. 530 Fairview Ave N Suite 2000 Seattle, Washington Tel: dxsupport@nanostring.com Molecules That Count Gene Expression Copy Number Variation Single Cell Gene Expression LBL-C

2 ncounter Elements TagSets General Purpose Reagents FOR LABORATORY USE. Intellectual Property Rights This ncounter Elements TagSets package insert and its contents are the property of NanoString Technologies, Inc. ( NanoString ) and are intended solely for the use of NanoString customers for the purpose of using ncounter Elements TagSets General Purpose Reagents. ncounter Elements TagSets and this package insert and any other documentation provided to you by NanoString in connection therewith are subject to patents, pending patents, copyright, trade secret rights, and other intellectual property rights owned by or licensed to NanoString. Trademarks NanoString, NanoString Technologies, ncounter, Molecules That Count, ndesign, and ncounter Elements 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 2014 NanoString Technologies, Inc. All rights reserved. Storage Conditions (TagSets) Document Information Version 04, created LBL-C

3 NanoString Technologies PACKAGE INSERT PREFACE... 4 Conventions Used... 4 Note Types... 4 Fonts... 4 Procedures... 4 Contact Information... 4 CHAPTER 1: Introduction Overview... 5 ncounter Elements TagSets Technology... 5 Materials Provided... 8 Contents Description... 8 Storage and Handling... 8 CHAPTER 2: Suggested RNA Hybridization Guidelines Overview... 9 Setting Up 12 Hybridization Reactions for RNA... 9 CHAPTER 3: Suggested DNA Hybridization Guidelines Overview...12 Fragmentation...12 CNV Reference Sample Considerations...12 Denaturation...13 DNA Input Amount...13 DNA Quality...13 Setting Up 12 Hybridization Reactions for DNA...14 CHAPTER 4: Creating Oligonucleotide Probe Pools Overview...16 Creating Master Probe Stocks...17 Creating 30X Working Probe Pools...18 SYMBOLS AND DEFINITIONS...19 Molecules That Count Gene Expression Copy Number Variation Single Cell Gene Expression 3

4 ncounter Elements TagSets General Purpose Reagents 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: NOTE IMPORTANT BOLD ITALICS This note type emphasizes general information. 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. When appearing in text or in a procedure, the bold text serves to highlight a specific action or refer to another step in the the instruction manual. Bold text is also used highlight important text or terms. Blue text is used to help the reader identify active hyperlinks. 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, or image important to facilitate clarity and understanding. NanoString Technologies, Inc. 530 Fairview Ave N Suite 2000 Seattle, Washington USA Tel: NANO (6266) Fax: dxsupport@nanostring.com 4

5 1 Introduction Overview This manual describes the recommended procedures for setting up hybridization reactions utilizing ncounter Elements TagSets General Purpose Reagents (GPRs). ncounter Elements TagSets are flexible, off-the-shelf reagents that when used in conjunction with other GPRs and ASRs can be used to develop assays for measuring digital counts of nucleic acid targets. They can be used to develop RNA-based assays (for example, gene expression or expressed fusion gene assays) or DNA-based assays (for example, Copy Number Variation or ChIP readout assays). Assays developed with Elements TagSets are compatible with a variety of sample sources including but not limited to: formalin-fixed paraffin-embedded (FFPE) tissue, fresh frozen tissue, blood products, fine-needle aspirates, cell lines, and immunoprecipitated nucleic acids. All ncounter Elements TagSets reagents are based on NanoString s core technology for measuring the abundance of nucleic acids via digital detection of fluorescent molecular barcodes. The TagSets described in this manual allow for detection of up to 216 unique target sequences. This manual describes in detail the recommended methods for setting up ncounter Elements TagSets hybridization reactions when working with RNA or DNA as well as considerations for ordering target-specific oligonucleotide probes. ncounter Elements TagSets Technology NanoString s ncounter Elements TagSets technology is based on molecular barcoding and digital quantification of target RNA or DNA sequences through the use of an ncounter Elements TagSet and target-specific oligonucleotide probe pairs (supplied by the user). The TagSet consists of fluorescently labeled specific Reporter Tags and a biotinylated universal Capture Tag (Figures 1.1 and 1.2). The Reporter Tags each have a unique pattern of six spots of color, creating fluorescent barcodes that can be individually resolved and counted during data collection. The universal Capture Tag enables hybridized complexes to be captured wtih streptavidin. Barcode 1 Tag T001 Barcode 2 Tag T002 Barcode 3 Tag T003 FIGURE 1.1: Examples of Reporter Tags with unique fluorescent barcodes and recognition sequences. Molecules That Count Gene Expression Copy Number Variation Single Cell Gene Expression 5

6 ncounter Elements TagSets General Purpose Reagents During hybridization, the specific Reporter Tags and universal Capture Tag hybridize to a pair of target-specific oligonucleotide probes (sourced by end-user, not provided by NanoString), which in turn hybridize directly to the single-stranded RNA or DNA target (Figure 1.2). Probe A hybridizes to a specific Reporter Tag and the 5 region of the target nucleic acid sequence. Probe B hybridizes to the universal Capture Tag and the 3 region of the target nucleic acid sequence. Each complete structure containing the target RNA or DNA, two oligonucleotide probes, and the Reporter and Capture Tags is referred to as a Tag Complex. Reporter Tag Selected probes procured by end user Universal Capture Tag A unique tag sequence Probe A** Probe B** is assigned to each target sequence 5 3 Nucleic Acid Target FIGURE 1.2: Customized oligonucleotide probes A and B (sourced by end-user, not provided by NanoString) hybridize with Reporter and Capture Tags, respectively, and the target nucleic acid to create a Tag Complex. The TagSet and probes are present in the reaction in large excess to the target molecules to ensure that each target undergoes hybridization. After hybridization, excess probes and tags as well as non-target nucleic acids may be washed away using a two-step magnetic bead-based purification process. The hybridized complex can then be captured via interaction of the universal Capture Tag (biotin) with streptavidin. The barcodes can then be imaged and counted. The CodeSet is comprised of the Elements TagSet and the corresponding oligonucleotide probes (supplied by the user) that recognize the targets of interest (Figure 1.3). Sixteen different core TagSets are available in increments of 12 tags. It is important to remember that larger TagSets include all tags found in smaller TagSets. For example, a core TagSet of 12 tags includes T001 through T012, and a core TagSet of 24 tags includes T001 through T024. The largest core TagSet currently available can detect up to 192 targets. Reporter Tag Probe A Capture Tag Probe B CodeSet FIGURE 1.3: Combining Elements TagSet general purpose reagents with user-supplied oligonucleotide probes produces a custom CodeSet for the detection of a specific set of targets. 6

7 NanoString Technologies PACKAGE INSERT Two core TagSets cannot be combined to increase the number of targets in an experiment as some of their Reporter Tags will overlap. To add additional targets to an ongoing experiment, use an Extension TagSet, which is available for 12 or 24 targets and does not include controls. The unique Reporter Tags in the Extension TagSets do not coincide with any of those found in the core TagSets. Assigning reference genes and other frequently analyzed targets to Reporter Tags with the lowest tag number will make the most efficient use of assay reagents in experiments where the target set is expected to evolve over the course of the project. In this way, the same oligonucleotide probes can be used for common targets in more than one experiment. There are multiple ways to examine additional targets in an assay: (1) order a larger TagSet, (2) order an Extension TagSet, or (3) order new oligonucleotide probes that link Reporter Tags in the existing TagSet with new targets. Core TagSet Core TagSet Core TagSet Extension TagSet Tag Target Tag Target Tag Target Tag Target T001 Gene A T001 Gene A T001 Gene A X001 Gene M T002 Gene B T002 Gene B T002 Gene B X002 Gene N T003 Gene C T003 Gene M T003 Gene C X003 Gene O T004 Gene D T004 Gene N T004 Gene D X004 Gene P T005 Gene E T005 Gene E T005 Gene E X005 Gene Q T006 Gene F T006 Gene F T006 Gene F X006 Gene R T007 Gene G T007 Gene G T007 Gene G X007 Gene S T008 Gene H T008 Gene O T008 Gene H X008 Gene T T009 Gene I T009 Gene P T009 Gene I X009 Gene U T010 Gene J T010 Gene Q T010 Gene J X010 Gene V T011 Gene K T011 Gene R T011 Gene K X011 Gene W T012 Gene L T012 Gene L T012 Gene L X012 Gene X FIGURE 1.4: An existing CodeSet can be modified by reassigning Reporter Tags to new targets or adding an extension TagSet. Targets that have been added or replaced are indicated in blue. Molecules That Count Gene Expression Copy Number Variation Single Cell Gene Expression 7

8 ncounter Elements TagSets General Purpose Reagents Materials Provided TagSets are available for analysis of 12 to 216 targets. Core TagSets are pre-mixed with a comprehensive set of controls and enable analysis of 12 to 192 targets depending on the product ordered. Extension TagSets, provided without controls, can be added to any core TagSet to expand the multiplexing capability by 12 or 24 targets. Each ncounter Elements TagSet is provided with sufficient material to perform 12 hybridization reactions. TABLE 1.1: Materials provided for ncounter Elements TagSets hybridization reactions Product Type Description Product Number ncounter Elements TagSet (Contains reagents for 12 reactions; includes controls) 12 Tags 24 Tags 36 Tags 48 Tags 60 Tags 72 Tags 84 Tags 96 Tags 108 Tags 120 Tags 132 Tags 144 Tags 156 Tags 168 Tags 180 Tags 192 Tags ELE-P1TS-012 ELE-P1TS-024 ELE-P1TS-036 ELE-P1TS-048 ELE-P1TS-060 ELE-P1TS-072 ELE-P1TS-084 ELE-P1TS-096 ELE-P1TS-108 ELE-P1TS-120 ELE-P1TS-132 ELE-P1TS-144 ELE-P1TS-156 ELE-P1TS-168 ELE-P1TS-180 ELE-P1TS-192 ncounter Elements Extension TagSet (Contains reagents for 12 reactions; no controls provided) 12 Tags 24 Tags ELE-P1EX-012 ELE-P1EX-024 Contents Description ncounter Elements TagSet 12 Reaction Aliquot (1 x 65 μl) buffer, nucleic acids, nucleic acids with fluorescent dyes. ncounter Elements Extension TagSet 12 Reaction Aliquot (1 x 70 μl) buffer, nucleic acids, nucleic acids with fluorescent dyes. Storage and Handling The expiration date for the ncounter Elements TagSet is listed on the outer box labeling. Reagents must be stored at -80 C. 8

9 2 Suggested RNA Hybridization Guidelines Overview ncounter Elements TagSets technology can be used to detect RNA for measuring gene expression. These reagents are compatible with a wide variety of sample types, including whole cell lysate as well as RNA purified from FFPE tissue, fresh frozen tissue, blood products, fine-needle aspirates, cell lines, and others. See below for recommended instructions for hybridizing the Reporter and Capture Tags, oligonucleotide probes, and RNA sample. IMPORTANT: Do not vortex or pipet vigorously as this may shear the Reporter Tags. Mix only by flicking or inverting tubes. A picofuge is preferable when spinning down solutions due to its low speed. If using a microfuge, do not pulse the machine as it may reach maximum speed and spin the TagSet out of solution. Spin at less than 1,000 rpm for no more than 30 seconds. NOTE: The following guidelines cover general steps for setting up RNA hybridization reactions. Each specific application using ncounter Elements TagSets GPRs will require optimization and validation using appropriate performance metrics defined by the end user. All reagents are provided for multiples of 12 reactions. Scale up the suggested volumes for additional reactions. Note that the instructions for 12 reactions use a multiplier of 13 to allow for dead volume in the master mix. Molecules That Count Gene Expression Copy Number Variation Single Cell Gene Expression 9

10 ncounter Elements TagSets General Purpose Reagents Each final hybridization reaction will have a volume of 30 μl and contain the following components: 10 μl of a hybridization buffer (EDTA and NaCl in phosphate buffer), 5 μl of TagSet, 1 μl of 30X Working Probe A Pool (0.6 nm each Probe A), 1 μl of 30X Working Probe B Pool (3 nm each Probe B), and up to 13 μl of sample RNA. If using an Extension TagSet, add 5 μl of the Extension TagSet, 1 μl of 30X Extension Probe A Pool and 1 μl of 30X Extension Probe B Pool and reduce the total volume of sample to no more than 6 μl (Figure 2.1). Instructions on preparing 30X Working Probe Pools are provided in Chapter 4. FIGURE 2.1: Guide to creating master mix and preparing RNA hybridization reactions. TagSet Hybridization Buffer 30X Working Probe A Pool 30X Working Probe B Pool Aliquot master mix to each tube Each Reaction 13 Reactions Hybridization Buffer 10 μl 130 μl TagSet 5 μl 65 μl 30X Working Probe A Pool 1 μl 13 μl 30X Working Probe B Pool 1 μl 13 μl Sample* Total Volume Up to 13 μl 30 μl Add sample RNA to each tube (see guidelines) Each Reaction 13 Reactions Add nuclease-free water, if needed, to reach a final volume of 30 μl Hybridization Buffer 10 μl 130 μl TagSet 5 μl 65 μl Extension TagSet 5 μl 65 μl 30X Working Probe A Pool 1 μl 13 μl Hybridization reactions at 67 C for at least 16 hours 30X Extension Probe A Pool 1 μl 13 μl 30X Working Probe B Pool 1 μl 13 μl 30X Extension Probe B Pool 1 μl 13 μl Sample* Total Volume Up to 6 μl 30 μl *NanoString recommends preparing samples with a high concentration to facilitate reducing the sample volume if an extension TagSet is later necessary. IMPORTANT: Master Probe Stocks must be diluted to 30X Working Probe Pools (see page 18). Addition of concentrated Master Probe Stocks directly to the hybridization reaction will have a severe adverse impact on results. 10

11 NanoString Technologies PACKAGE INSERT 1. Follow the directions in Chapter 4 to create the 30X Working Probe Pools. The accuracy of the probe concentration in the hybridization reaction is important for maximizing sensitivity. Be sure to dilute the pools to the appropriate 30X concentration before addition to the master mix below. 2. If the samples contain total RNA, go to Step If the samples will be cell or tissue lysates, prepare them according to the recommended instructions in your supplier s kit. a. Cells or tissues should be lysed at an appropriate concentration so that no more than 3 μl of lysis buffer is added to the final 30 μl reaction. Larger amounts of lysis buffer will impact the melting temperature of the probes during hybridization and lead to lower sensitivity. For reference, 10,000 mammalian cells yield approximately 100 ng of total RNA. b. Lysates should be aliquoted and stored at -80 C. Avoid freeze/thaw cycles. 4. Remove an aliquot of the TagSet from storage at -80 C and thaw it on ice. Invert several times to mix well, and briefly spin down the reagent at less than 1,000 rpm. 5. Each core TagSet tube contains 65 μl of reagent. Create a master mix by adding reagents directly into the TagSet tube. First add 130 μl of hybridization buffer, followed by 13 μl of the 30X Working Probe A Pool. Mix well by flicking the tube and briefly spin down at less than 1,000 rpm before adding 13 μl of the 30X Working Probe B Pool. Mix and spin again. a. If using an Extension TagSet, also add 65 μl of the Extension TagSet reagent and 13 μl of the 30X Extension Probe A Pool before mixing. Then also add 13 μl of the 30X Extension Probe B Pool before the final mixing step. b. If all samples have the same volume, additional nuclease-free water may be added to the master mix now instead of to individual tubes during Step 9. Increase the aliquot in Step 7 as necessary. 6. Label a 12-tube strip and cut it in half so it will fit in a picofuge. 7. Add 17 μl (core TagSet only) or 24 μl (core TagSet plus Extension TagSet) of master mix to each of the 12 tubes using a fresh pipette tip for each well. 8. Add RNA according to the sample type as follows: a. If using total RNA: Add total RNA sample (maximum volume of 13 μl for core TagSet only, or 6 μl for core TagSet plus Extension TagSet) to each tube. NanoString recommends starting with ng of total RNA per hybridization reaction and performing a titration to determine the optimal lower limit. b. If using cell lysates: Add cell lysate sample (maximum volume of 3 μl) to each tube. NanoString recommends starting with 5,000-10,000 cells per hybridization reaction and performing a titration to determine the optimal lower limit. IMPORTANT: If using cell lysates, adding more than 3 μl of sample will have an adverse impact on hybridization efficiency of some probes due to the denaturing effect of the lysis buffer. Use the same amount of lysate when comparing samples. 9. If necessary, add nuclease-free water to each tube to bring the volume of each reaction to 30 µl. 10. Program the thermal cycler to use a 30 μl volume, calculated temperature, and heated lid. Set at 67 C for the selected hybridization time (typically hours; see Step 11) and then ramp down to 4 C. To minimize the potential for evaporation, the thermal cycler lid should be set at 5 C above the block temperature. Cap tubes and mix the reagents by inverting the strip tubes several times and flicking with a finger to ensure complete mixing. Briefly spin down the hybridization reactions at less than 1,000 rpm and immediately place the strip tubes in the thermal cycler. 11. Incubate reactions for at least 16 hours but no more than 48 hours. Ramp reactions down to 4 C when complete and process the following day. Do not leave the reactions at 4 C for more than 24 hours or increased background may result. NOTE: The purpose of selecting a fixed hybridization time followed by a ramp down to 4 C is to ensure equivalent hybridization times of all assays being directly compared in the same series of experiments. Hybridization efficiency improves with time, and target counts may increase 5% per hour between 16 and 24 hours. Although a 16-hour incubation is adequate for most purposes, a longer incubation will typically increase counts. Molecules That Count Gene Expression Copy Number Variation Single Cell Gene Expression 11

12 ncounter Elements TagSets General Purpose Reagents 3 Suggested DNA Hybridization Guidelines Overview ncounter Elements TagSets technology can be used to detect DNA for purposes such as determining copy number variation (CNV) and performing counts of genetic loci in enriched DNA. Reagents are compatible with a wide variety of DNA sample types, including formalinfixed paraffin-embedded (FFPE) tissue, fresh frozen tissue, blood products, fine-needle aspirates, cell lines, and immunoprecipitated DNA. However, additional sample processing steps are required before assaying most types of DNA samples. Read these important considerations before performing the hybridization protocol if using ncounter Elements TagSet reagents with DNA samples. Fragmentation Purified genomic DNA is typically larger in size (> 20 kb) than the average mrna (~1.5 kb). To ensure adequate hybridization kinetics, DNA must be fragmented prior to hybridization and analysis. In the case of immunoprecipitated material (ChIP-string assays), the DNA has typically been fragmented during the IP process and additional fragmentation is not required. Two methods of fragmentation are recommended: digestion by the Alu1 restriction enzyme, or Covaris AFA -based fragmentation. When using high molecular weight genomic DNA from cell lines, blood, or fresh or frozen tissue, NanoString recommends using Alu1-based fragmentation. Digestion of human genomic DNA with Alu1 results in an average fragment size of 500 bases. Optimal target fragments for hybridization (~ bases) are selected during the probe design process. When using degraded genomic DNA (e.g., from FFPE samples) NanoString recommends using Covaris AFA -based fragmentation, although Alu1 can also be used. The optimal target size is bases. It may also be possible to use standard sonication or chemical or mechanical fragmentation methods if fragment length is controlled adequately. For best results, all samples (including the reference sample) should have similar fragmentation profiles. CNV Reference Sample Considerations Genomic DNA from a reference sample may be used to determine copy number with ncounter Elements TagSets. Reference samples should not contain copy number variations in the test regions. For best results, select one or more genomic DNA reference samples that are of the same sample type as the test samples (e.g., FFPE, cell line, or blood) and have been purified and fragmented in the same way as the test samples. The reference DNA(s) should be run at least once whenever the content of the CodeSet is changed (i.e., different loci are being measured). Please review detailed guidelines and suggested protocols for fragmenting DNA samples in the ncounter DNA Assay User Manual. 12

13 NanoString Technologies PACKAGE INSERT Denaturation DNA is typically double-stranded in its biological conformation, whereas RNA is single-stranded. Double-stranded DNA must be denatured prior to hybridization. IMPORTANT: NanoString recommends a denaturation temperature of 95 C for 5 minutes, followed by an immediate ramp down to 4 C or quick cooling on ice. Incomplete denaturation results in decreased sensitivity. DNA Input Amount The recommended mass of input genomic DNA for most hybridization reactions is ng, three times greater than the mass of input RNA recommended as a starting point when measuring gene expression. This is due to the fact that most non-repetitive genomic DNA sequences are present at two copies per cell in normal diploid samples. In contrast, the amount of a given RNA can range from one to many thousands of copies per cell. The maximum sample input volume for DNA hybridization is 13 μl using a core TagSet, and 6 μl if an Extension TagSet is added. Thus, the sample DNA concentration must be high enough to provide an adequate mass. If the available sample is not a limiting factor, increasing the DNA input amount to 600 ng may provide better resolution for small variations in CNV assays and increase accuracy for highly degraded FFPE samples. For DNA that has been enriched (e.g., via immunoprecipitation, exome capture, or target enrichment) the input requirements are typically lower than those required for standard genomic DNA. For these sample types, recommended starting input amounts are 5-50 ng depending on the level of enrichment. Input amounts may need to be optimized for some experimental conditions. DNA Quality DNA should be free of contaminating RNA for accurate copy number analysis. NanoString strongly recommends that DNA preparations be RNAse-treated as described by the extraction kit manufacturer. RNA contamination may negatively impact the quality of the data in two ways: 1. Even when probes are selected to non-coding sequences, the presence of unprocessed RNA may result in artificially increased signal and copy number determinations. 2. RNA may result in over-estimation of DNA concentration when measured by UV absorbance, leading to lower than recommended DNA input amounts and lower counts. For pure genomic DNA preparations, NanoString recommends A260/280 ratios between 1.7 and 1.9 and A260/230 ratios between 1.3 and 2.0. Fluorescence-based assays using dyes specific for DNA may provide the most accurate concentration measurements if RNA contamination is suspected. DNA extracted from FFPE samples is typically degraded due to the fixation and storage process. NanoString recommends that the average size of extracted DNA be greater than 1 kb prior to fragmentation. Molecules That Count Gene Expression Copy Number Variation Single Cell Gene Expression 13

14 ncounter Elements TagSets General Purpose Reagents IMPORTANT: Do not vortex or pipet vigorously as this may shear the Reporter Tags. Mix only by flicking or inverting tubes. A picofuge is preferable when spinning down solutions due to its low speed. If using a microfuge, do not pulse the machine as it may reach maximum speed and spin the TagSet out of solution. Spin at less than 1,000 rpm for no more than 30 seconds. NOTE: The following guidelines cover general steps for setting up DNA hybridization reactions. Each specific application using ncounter Elements TagSets GPRs will require optimization and validation using appropriate performance metrics defined by the end user. All reagents are provided for multiples of 12 reactions. Scale up the suggested volumes for additional reactions. Keep in mind that these instructions for 12 reactions use a multiplier of 13 to allow for one reaction s worth of dead volume in the master mix. The final hybridization reaction will have a volume of 30 μl and contain the following components in each tube: 10 μl of a hybridization buffer (EDTA and NaCl in phosphate buffer), 5 μl of TagSet, 1 μl of 30X Working Probe A Pool (0.6 nm each Probe A), 1μL of 30X Working Probe B Pool (3 nm each Probe B), and up to 13 μl of sample DNA (Figure 3.1). If using an Extension TagSet, also add 5 μl of the Extension TagSet, 1 μl of the 30X Extension Probe A Pool and 1 μl of 30X Extension Probe B Pool, and reduce the total volume of sample to no more than 6 μl (Figure 3.1). Instructions on preparing 30X Working Probe Pools are provided in Chapter 4. FIGURE 3.1: Guide to creating master mix and preparing DNA hybridization reactions. TagSet Hybridization Buffer 30X Working Probe A Pool 30X Working Probe B Pool Aliquot master mix to each tube Each Reaction 13 Reactions Hybridization Buffer 10 μl 130 μl TagSet 5 μl 65 μl 30X Working Probe A Pool 1 μl 13 μl 30X Working Probe B Pool 1 μl 13 μl Sample* Total Volume Up to 13 μl 30 μl Add sample DNA to each tube (see guidelines) Each Reaction 13 Reactions Add nuclease-free water, if needed, to reach a final volume of 30 μl Hybridization Buffer 10 μl 130 μl TagSet 5 μl 65 μl Extension TagSet 5 μl 65 μl 30X Working Probe A Pool 1 μl 13 μl Hybridization reactions at 67 C for at least 16 hours 30X Extension Probe A Pool 1 μl 13 μl 30X Working Probe B Pool 1 μl 13 μl IMPORTANT: Master Probe Stocks must be diluted to 30X Working Probe Pools (see page 18). Addition of concentrated Master Probe Stocks directly to the hybridization reaction will have a severe adverse impact on results. 30X Extension Probe B Pool 1 μl 13 μl Sample* Total Volume Up to 6 μl 30 μl *NanoString recommends preparing samples with a high concentration to facilitate reducing the sample volume if an extension TagSet is later necessary. 14

15 NanoString Technologies PACKAGE INSERT 1. See the directions in Chapter 4 to create the 30X Working Probe Pools. The accuracy of the probe concentration in the hybridization reaction is important for maximizing sensitivity. Be sure to dilute the pools to the appropriate 30X concentration before addition to the master mix below. 2. Remove an aliquot of the TagSet from storage at -80 C and thaw it on ice. Invert several times to mix well, and briefly spin down the reagent at less than 1,000 rpm. 3. Each TagSet tube contains 65 μl of reagent. Create a master mix by adding reagents directly into the TagSet tube. First add 130 μl of hybridization buffer, followed by 13 μl of the 30X Working Probe A Pool. Mix well by flicking the tube and briefly spin down at less than 1,000 rpm before adding 13 μl of the 30X Working Probe B Pool. Mix and spin again. a. If using an Extension TagSet, also add 65 μl of the Extension TagSet reagent and 13 μl of the 30X Extension Probe A Pool before mixing. Then also add 13 μl of the 30X Extension Probe B Pool before the final mixing step. b. If all samples have the same volume, additional nuclease-free water may be added to the master mix now instead of to individual tubes during Step 8. Increase the aliquot in Step 5 as necessary. 4. Label a 12-tube strip and cut it in half so it will fit in a picofuge. 5. Add 17 μl (core TagSet only) or 24 μl (core TagSet plus Extension TagSet) of master mix to each of the 12 tubes using a fresh pipette tip for each well. 6. Denature DNA samples at 95 C for 5 minutes, followed by an immediate ramp down to 4 C or quick cooling on ice. 7. Add fragmented and denatured DNA sample (maximum volume of 13 μl for core TagSet only, or 6 μl for core TagSet plus Extension TagSet) to each tube. See the above sections titled Fragmentation and DNA Input Amount for guidelines. If the sample was isolated by ChIP, fragmentation is not necessary 8. If necessary, add nuclease-free water to each tube to bring the volume of each reaction to 30 μl. 9. Program the thermal cycler to use a 30 μl volume, calculated temperature, and heated lid. Set at 67 C for the selected hybridization time (typically hours; see Step 10) and then ramp down to 4 C. To minimize the potential for evaporation, the thermal cycler lid should be set at 5 C above the block temperature. Cap tubes and mix the reagents by inverting the strip tubes several times and flicking with a finger to ensure complete mixing. Briefly spin down the hybridization reactions at less than 1,000 rpm and immediately place the strip tubes in the thermal cycler. 10. Incubate reactions for at least 16 hours but no more than 48 hours. Ramp reactions down to 4 C when complete and process the following day. Do not leave the reactions at 4 C for more than 24 hours or increased background may result. NOTE: The purpose of selecting a fixed hybridization time followed by a ramp down to 4 C is to ensure equivalent hybridization times of all assays being directly compared in the same series of experiments. Hybridization efficiency improves with time, and target counts may increase 5% per hour between 16 and 24 hours. Although a 16-hour incubation is adequate for most purposes, a longer incubation will typically increase counts. Molecules That Count Gene Expression Copy Number Variation Single Cell Gene Expression 15

16 ncounter Elements TagSets General Purpose Reagents 4 Creating Oligonucleotide Probe Pools Overview The oligonucleotide probes must be formatted into two separate pools, one containing all Probe A s (Master Probe A Stock), and one containing all Probe B s (Master Probe B Stock). If stored in aliquots at -20 C to -80 C, the Master Probe Stocks can be used for many experiments. Please refer to the oligonucleotide supplier for specific storage recommendations and shelf-life information. Before setting up the hybridization reactions, 30X Working Probe Pools must be generated by diluting each Master Probe Stock. The final concentration of the oligonucleotides in 30X Working Probe A Pool will be 0.6 nm each, and the final concentration of the oligonucleotides in 30X Working Probe B Pool will be 3 nm each. The absolute concentration of the pool will vary depending on the plex of the experiment; due to the dilute DNA concentrations of many 30X Working Probe Pools, long-term storage and reuse of these pools is not recommended. The protocols below provide an example of how to generate the Master Probe Stocks and 30X Working Pools for Probe A and Probe B. Depending on the oligonucleotide format obtained from the supplier, different pipetting volumes and dilutions may be necessary to achieve the required final concentrations. NOTE: The following guidelines cover general steps for use with ncounter Elements TagSets. Each specific application using ncounter Elements TagSets will require optimization and validation using appropriate performance metrics defined by the end user. IMPORTANT: The concentrations of Probe A and Probe B in the hybridization reaction are critical for maximizing the sensitivity of the assay. Be sure to follow appropriate pooling and dilution protocols carefully to create accurate 30X Working Probe Pools. 16

17 NanoString Technologies PACKAGE INSERT Creating Master Probe Stocks NOTE: Some oligo suppliers will provide pooled oligos which can be used in place of creating your own Master Probe Stocks. If utilizing this service, specify a pool of Probe A s at a final concentration of 5 nm per oligo, and a separate pool of Probe B s at a final concentration of 25 nm per oligo. Pooled probes provided by oligo suppliers at the recommended concentrations must still be further diluted to create 30X Working Probe Pools (see next page) before addition to the assay. IMPORTANT: Always create separate Probe A and Probe B stocks. Do NOT create a combined Master Probe Stock containing Probe A s and Probe B s in the same tube; elevated background and lowered assay sensitivity may result. 1. NanoString recommends resuspending individual Probe A oligonucleotides at a 1 μm concentration, and individual Probe B oligonucleotides at a 5 μm concentration. Oligonucleotides should be resuspended in TE (10 mm Tris ph 8, 1 mm EDTA) or a similar buffer and stored frozen under conditions recommended by the supplier. To prepare Master Probe Stocks, begin by removing the appropriate Probe A and Probe B oligonucleotides from storage at -80 C or -20 C and thawing them on ice. 2. Master Probe A Stock a. Pipet 5 μl of each Probe A (starting concentration 1 μm) into a 1.7 ml microfuge tube. b. Add TE to a final combined volume of 1 ml. c. The final concentration of each Probe A in the Master Probe A Stock will be 5nM. d. Store in aliquots at -20 C or -80 C as recommended by the supplier. 3. Master Probe B Stock a. Pipet 5 μl of each Probe B (starting concentration 5 μm) into a 1.7 ml microfuge tube. b. Add TE to a final combined volume of 1 ml. c. The final concentration of each Probe B in the Master Probe B Stock will be 25nM. d. Store in aliquots at -20 C or -80 C as recommended by the supplier. NOTE: The probes in the Master Probe Stocks must be appropriate for the targets being queried. If reporter tags are reassigned to new targets, new Master Probe Stocks containing the specific set of appropriate probes must be created. Do NOT add additional probes to existing Master Probe Stocks. If using an Extension Tagset as well as a Core TagSet, create separate Master Probe Stocks for the Extension Probes. IMPORTANT: Minimize freeze-thaw cycles by storing Master Probe Stocks in appropriate aliquots at -20 C or -80 C. Thaw each aliquot only once and then place at 4 C for use in creating multiple 30X Working Probe Pools. A suitable aliquot size for the workflow can be calculated from the information in Table 4.1 (see page 18) based on your expected assay throughput. Follow the supplier s guidance on stability of the oligonucleotide stocks at 4 C. Molecules That Count Gene Expression Copy Number Variation Single Cell Gene Expression 17

18 ncounter Elements TagSets General Purpose Reagents Creating 30X Working Probe Pools IMPORTANT: Always create separate Probe A and Probe B 30X Working Probe Pools. Do NOT create a combined 30X Working Probe Pool containing Probe A s and Probe B s in the same tube; elevated background and lowered assay sensitivity may result X Working Probe A Pool a. Determine the number of assays to be performed. Starting with the Master Probe A Stock, follow the 8.3-fold dilution outlined in Table 4.1 to generate a 30X Working Probe A Pool at the appropriate scale. b. Mix well and spin to bring contents to the bottom of the tube. The final concentration of each Probe A in the 30X Working Probe A Pool will be 0.6 nm. c. Proceed to the Hybridization Guidelines for RNA or DNA described in Chapters 2 and 3. The final concentration of each Probe A in the 30 μl hybridization assay will be 20 pm. d. Due to the dilute DNA concentrations of many 30X Working Probe Pools, long-term storage and reuse of these pools is not recommended. A fresh dilution of the Master Probe Stock should be made for subsequent assays X Working Probe B Pool a. Determine the number of assays to be performed. Starting with the Master Probe B Stock, follow the 8.3-fold dilution outlined in Table 4.1 to generate a 30X Working Probe B Pool at the appropriate scale. b. Mix well and spin to bring contents to the bottom of the tube. The final concentration of each Probe B in the 30X Working Probe B Pool will be 3 nm. c. Proceed to the Hybridization Guidelines for RNA or DNA described in Chapters 2 and 3. The final concentration of each Probe B in the 30 μl hybridization assay will be 100 pm. d. Due to the dilute DNA concentrations of many 30X Working Probe Pools, long-term storage and reuse of these pools is not recommended. A fresh dilution of the Master Probe Stock should be made for subsequent assays. TABLE 4.1: Guide to diluting Master Probe Stocks to generate 30X Working Probe Pools. Due to the dilute nature of the final pool, use of TE-Tween (10 mm Tris ph 8, 1 mm EDTA, 0.1% Tween -20) is recommended. Number of Assays Aliquot from Master Probe Stock (μl) TE-Tween (μl) Final Volume (μl)

19 ncounter Elements TagSets General Purpose Reagents Manufacturer Consult Instructions for Use Catalogue or Reference Number Batch Code / Lot Number Temperature Range Storage Conditions Lower Limit of Temperature Storage Conditions Upper Limit of Temperature Storage Conditions For Use By / Expiry Date Date of Manufacture Room Temp. = Room Temperature HYB = Hybridization Regulatory Disclaimer FOR LABORATORY USE NanoString Technologies, Inc. All rights reserved. NanoString, NanoString Technologies, ncounter, Molecules That Count, ndesign, and ncounter Elements are registered trademarks or trademarks of NanoString Technologies, Inc., ( NanoString ) in the United States and/or other countries. Changes to Previous Versions Version Number LBL-C LBL-C LBL-C LBL-C Description Initial release Adjusted probe-pooling guidance Adjusted guidance for performing hybridizations with RNA lysates and added section on verifying oligonucleotide probes Adjusted guidance for performing hybridizations and pooling probes and removed section on verifying oligonucleotide probes 19

20 ncounter Elements TagSets General Purpose Reagents NanoString Technologies, Inc. CONTACT US SALES CONTACTS 530 Fairview Ave N Suite 2000 Seattle, Washington dxsupport@nanostring.com United States: us.sales@nanostring.com Tel: (888) Europe: europe.sales@nanostring.com Fax: (206) Other Regions: info@nanostring.com NanoString Technologies, Inc. All rights reserved. NanoString, NanoString Technologies, ncounter, Molecules That Count, ndesign, and ncounter Elements 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. The manufacture, use and/or sale of NanoString product(s) may be subject to one or more patents or pending patent applications owned by NanoString or licensed to NanoString from Life Technologies Corporation and other third parties. FOR LABORATORY USE. LBL-C