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1. Station the next cartridge is ready to be loaded Processed cartridges can be loaded onto the Digital Analyzer sequentially even while an other cartridge is being imaged Literature Cited Amit I Garber M Chevrier N Leite A P Donner Y Eisenhaure T Guttmann M Grenier J K Li W Zuk O Schubert L A Birditt B Shay T Goren A Zhang X Smith Z Deering R McDonald R C Cabili M Bernstein B E Rinn J L Meissner A Root D E Hacohen N and Regev A 2009 Unbiased reconstruction of a mammalian tran scriptional network mediating pathogen re sponses Science 326 257 263 Burczynski M E Peterson R L Twine N C Zuberek K A Brodeur B J Casciotti L Maganti V Reddy P S Strahs A Immer mann F Spinelli W Schwertschlag U Slager A M Cotreau M M and Dorner A J 2006 Molecular classification of Crohn s dis ease and ulcerative colitis patients using tran scriptional profiles in peripheral blood mononu clear cells J Mol Diagn 8 51 61 Devonshire A S Elaswarapu R and Foy C A 2010 Evaluation of external RNA con trols for the standardisation of gene expres sion biomarker measurements BMC Genomics 11 662 Evans S J Datson N A Kabbaj M Thompson R C Vreugdenhil E De Kloet E R Watson S J and Akil H 2002 Evaluation of Affymetrix Gene Chip sensitivity in rat hip pocampal tissue using SAGE analysis Serial analys2. The twelve RCC data files corresponding to all samples in the cartridge are combined in an RCC zip folder At 600 FOV the analyzer takes approximately 4 5 hr to acquire images for all twelve flow cells of a sample cartridge Select Download Data on the touch screen to download the RCC zip folder containing all RCC data files to the memory stick Analyze data 12 13 14 15 Using Excel import the individual RCC data files into the RCC collector file template provided on the memory stick Open the RCC collector file in Excel enable the use of macros by selecting the Enable this content radio button and then click the Import RCC Files button All RCC data files that need be analyzed together should be present in the same folder location to multi select the files and have the same RLF associated with them If the abundance of target mRNA molecules is going to be compared across multiple different samples normalize the counts for all target genes in all samples flow cells based on the positive spike in controls to account for differences in hybridization effi ciency and post hybridization processing including purification and immobilization of complexes Sum the counts for the positive spike in controls for each sample flow cell to estimate the overall assay efficiency Then calculate a normalization factor for each sample based on the relative number of positive control counts For each sample flow cell multiply
3. http www luminexcorp com offers high throughput sig nature gene expression analysis for up to 100 different transcripts across many samples Landegren et al 1988 Hsuih et al 1996 Nilsson et al 2000 However this assay has a much lower sensitivity than the nCounter system more comparable to microarrays and requires enzymatic conversion of mRNA to cDNA followed by amplification Peck et al 2006 In general Affymetrix GeneChips the gold standard in microarray technology can detect a minimum of 10 mRNA copies cell with good reproducibility Evans et al 2002 There are many applications for this tech nology including detection of low abundance subtracted counts 0 1 10 Concentration fM 25000 er R2 0 9786 S2R 0 a J O O O O _ _ Q o O J O pa O X O OQ 0 20 60 80 Concentration fM B a i 22 10000 R 0 9803 S2R _INS40 transcripts that cannot be detected by mi croarrays Other applications include high throughput screening small molecule or RNA interference using gene expression signature based read outs chromatin im munoprecipitation assays splice variation analysis pathway analysis fusion transcript expression and pathogen identification Amit et al 2009 In principle the assay can be used to measure other types of nucleic acids includ ing microRNAs and long non coding RNAs Critical Parameters and Troublesho
4. set of genes whose pattern of expression can be used as readout for the process under study This can be achieved by two general methods Gene sets that accurately reflect a particular biological process can be determined empirically by genome wide expression profiling using either microarrays or transcriptome sequencing by RNA Seq UNIT 4 11 under conditions where the biological process of interest is activated For example gene expression signatures that reflect activation of the insulin signaling pathway can be generated using RNA Seq to measure genome wide expression changes induced in cells stimulated with insulin at different time points These gene expression data can then be used to identify a pattern of gene expression a signature that reflects the phenotype of insulin pathway activation Amit et al 2009 identified 118 signature genes that define the response of mouse primary dendritic cells to infection by pathogens using microarray expression profiling of dendritic cells exposed to different pathogen derived components For details of the statistical method developed to identify informative signature genes in this system see Amit et al 2009 For a general review of standard statistical methods applied to microarray data to derive gene expression signatures see Nevins and Potti 2007 Once a robust gene expression signature representative of the biological process of interest is derived one can interrogate pharmacological and geneti
5. 800 reporter capture probe pairs in a single reaction it is advisable to incorporate at least three independent probe pairs per target mRNA in the CodeSet design Current Protocols in Molecular Biology Gene expression assays employ one or two reference genes to normalize transcript levels between different samples in order to account for differences in the amount of total RNA present in the samples While these reference genes are often housekeeping genes their expression levels frequently vary among tissues or cells and may change under different conditions and treatments Thus the selection of appropriate reference gene s is critical for the success of gene expression studies For this purpose at least ten reference genes that do not show changes in expression levels based on initial microarray analysis performed to select signature genes should be chosen It is preferable that the expression levels of the selected reference genes also span the dynamic range of the nCounter Gene Expression Assay low medium and high expression levels The assay has a linear dynamic range of 7 x 10 counts Positive spike in controls target sequences and corresponding reporter capture probe pairs are included in each CodeSet Targets for the positive spike in controls are 100 base oligonucleotides complementary to the spike in reporter and capture probes and are included in the reactions at final concentrations ranging from 0 1 to 100 fM The specifi
6. Cell 114 323 334 Lamb J Crawford E D Peck D Modell J W Blat I C Wrobel M J Lerner J Brunet J P Subramanian A Ross K N Reich M Hieronymus H Wei G Armstrong S A Haggarty S J Clemons P A Wei R Carr S A Lander E S and Golub T R 2006 The Connectivity Map Using gene expression sig natures to connect small molecules genes and disease Science 313 1929 1935 Landegren U Kaiser R Sanders J and Hood L 1988 A ligase mediated gene detection tech nique Science 241 1077 1080 Major M B Roberts B S Berndt J D Marine S Anastas J Chung N Ferrer M Yi X Stoick Cooper C L von Haller P D Kate gaya L Chien A Angers S MacCoss M Cleary M A Arthur W T and Moon R T 2008 New regulators of Wnt beta catenin sig naling revealed by integrative molecular screen ing Sci Signal 11 ra12 Nevins J R and Potti A 2007 Mining gene ex pression profiles Expression signatures as can cer phenotypes Nat Rev Genet 8 601 609 Nilsson M Barbany G Antson D O Gertow K and Landegren U 2000 Enhanced detec tion and distinction of RNA by enzymatic probe ligation Nat Biotechnol 18 791 793 Peck D Crawford E D Ross K N Stegmaier K Golub T R and Lamb J 2006 A method for high throughput gene expression signature analysis Genome Biol 7 R61 Rhodes D R and Chinnaiyan A M 2005 Integra tive analysis
7. The entire procedure can be divided into four major sections 1 hybridization of target mRNA to gene specific probe pairs 2 fully automated post hybridization processing 3 data acquisition imaging and processing of barcodes and 4 data analysis Here a brief overview of the protocol and underlying principles is provided followed by detailed discussions of the gene signature CodeSet controls and sample preparation Overview of the nCounter Gene Expression Assay A multiplexed CodeSet is assembled with two sequence specific probes for each target gene of interest Fig 25B 10 1A The capture probe contains from 5 to 3 a 35 to 50 base sequence complementary to a particular target mRNA a short sequence contain ing two repeats of a 15 base sequence common to all capture probes orange segment in Fig 25B 10 1A and a biotin affinity tag The reporter probe contains from 3 to 5 a second 35 to 50 base sequence complementary to the same target mRNA near or con tiguous with the target specific sequence in the capture probe partner a short sequence containing four repeats of a 15 base sequence common to all reporter probes black seg ment in Fig 25B 10 1A and a color coded molecular barcode The common sequences included in all capture and reporter probes facilitate the removal of unbound excess probes during post hybridization processing The barcode is composed of a linearized single stranded M13 DNA molecule annea
8. biological expression levels to minimize the contribution of noise inherent in hybridization based assays across multiple experiments Design and Synthesis of CodeSet Once a set of genes comprising the signature of interest has been established the following four steps achieve the design and synthesis of gene specific reporter and capture probes that will define the CodeSet 1 The RefSeq NCBI Reference Sequence database accession numbers for the mRNA transcripts of the selected signature genes is submitted to NanoString using the CodeSet Design Form 2 NanoString designs reporter and capture probe pairs for every gene in the signature gene list provided by the user The NCBI Reference Sequence database 1s used as the source for the target sequences within each reporter and capture probe To prevent off target effects due to cross hybridization of reporter capture probe pairs to non target molecules in the sample the full non redundant dataset containing the sequence information for a species from RefSeq 1s used to check the cross hybridizing potential of the target specific probe sequences Probe pairs that target most if not all of the transcript variants for a particular gene are selected by default Probes are screened for all standard hybridization parameters such as hybridization efficiency cross hybridization potential GC content and predicted secondary structures The best scoring reporter capture probe pair for each gene in the sign
9. comprising target mRNA bound to specific reporter capture probe pairs are isolated The biotin label at the 3 end of the capture probes is used to attach the complexes to streptavidin coated slides An electric field 1s applied to orient and extend the tripartite complexes on the surface of the slide to facilitate imaging and detection of the color coded molecules A microscope objective and a CCD camera are used to image the immobilized complexes The number of molecules for a particular mRNA species is counted by decoding the unique pattern of the fluorescent colors encoded in each reporter probe The protocol is performed from start to finish on the nCounter System which Current Protocols in Molecular Biology is designed to provide hybridization post hybridization processing and digital data acquisition capabilities in one simple workflow The integrated system is composed of two instruments the fully automated nCounter Prep Station for post hybridization processing and the Digital Analyzer for imaging data collection and data processing The CodeSet containing the reporter and capture probes targeting the genes of interest as defined by the user and all the reagents and consumables needed to perform the analysis are provided ready to use STRATEGIC PLANNING The nCounter assay can be used to detect many types of nucleic acids including mRNA DNA and microRNA This unit describes the protocol for measuring mRNA levels Fig 25B 10 1
10. it is likely that the level of reference gene mRNA may change under certain conditions The positive spike control counts can also be used for normal ization instead of a reference gene If data are not reproducible experiments to be compared should be set up on the same day and run in parallel Anticipated Results The limit of detection for this assay is as low as 0 5 fM based on spike in controls roughly corresponding to 1 copy per cell and is achieved 90 of the time Fig 25B 10 2 and supplementary file at http www currentprotocols com protocol mb25B10 The assay offers gt 1 5 fold change sensi tivity for target mRNAs with gt 5 copies per cell and gt 2 0 fold change sensitivity for low abundance mRNAs 1 copy per cell The assay also has a high degree of reproducibility with R gt 0 95 Fig 25B 10 3 and supplementary file with a linear dynamic range of 7 x 10 total counts S2Rt Linear S2R R2 0 99986 Normalized counts S2Rt_1 La on E Td on a 3 o D i i MH F E id S2Rt_INS40 S2Rt _INS40 Linear S2Rt _INS40 R 0 99983 03 C sr A agtt 1000 Normalized counts S2R _INS40_1 Figure 25B 10 3 Reproducibility of the nCounter platform A Scatter plot of normalized background subtracted counts for all 100 genes assayed shown in log scale for technical repli cates of baseline S2R samples S2R 1 vs S2R 2 Genes were not filtered base
11. of the cancer transcriptome Nat Genet 37 S31 S37 Rhodes D R Yu J Shanker K Deshpande N Varambally R Ghosh D Barrette T Pandey A and Chinnaiyan A M 2004 Large scale meta analysis of cancer microarray data identi fies common transcriptional profiles of neoplas tic transformation and progression Proc Natl Acad Sci U S A 101 9309 93 14 Current Protocols in Molecular Biology Rhodes D R Kalyana Sundaram S Mahavisno V Barrette T R Ghosh D and Chinnatyan A M 2005 Mining for regulatory programs in the cancer transcriptome Nat Genet 37 579 583 Spurgeon S L Jones R C and Ramakrishnan R 2008 High throughput gene expression mea surement with real time PCR in a microfluidic dynamic array PLoSONE 3 e1662 Subramanian A Tamayo P Mootha V K Mukherjee S Ebert B L Gillette M A Paulovich A Pomeroy S L Golub T R Lander E S and Mesirov J P 2005 Gene set enrichment analysis A knowledge based ap proach for interpreting genome wide expres sion profiles Proc Natl Acad Sci U S A 102 15545 15550 van de Vijver M J He Y D van t Veer L J Dai H Hart A A Voskuil D W Schreiber G J Peterse J L Roberts C Marton M J Parrish M Atsma D Witteveen A Glas A Delahaye L van der Velde T Bartelink H Rodenhuis S Rutgers E T Friend S H and Current Protocols in Molecular Biology Bernards R 2002
12. sample regardless of the level of multiplexing The disadvantage of the nCounter assay is its dependence on specialized equipment and the up front cost of setting up the technology The instrumentation Prep Station and Dig ital Analyzer costs approximately 230 000 U S and would ideally be bought and set up in a core facility rather than individual labs The price per data point for custom Code Sets decreases as the numbers of targets and samples increase thus making it economical for querying large sets of targets against large sample sets For example the price per data point for an average project including all nec essary reagents can range from 1 00 for a study of 100 genes in 96 samples with a single replicate to approximately 0 30 for a study of 400 genes in 384 samples in triplicate The BioMark real time quantitative PCR system Fluidigm Attp wwwfluidigm com is ahigh throughput system that uses microflu idics and a microfabricated device to perform Discovery of Differentially Expressed Genes 25B 10 13 Supplement 94 gt U Normalized background Average normalized thousands of real time quantitative PCR re actions simultaneously Golden et al 2008 Major et al 2008 Spurgeon et al 2008 Although the sensitivity achieved by the two platforms is comparable the Fluidigm sys tem does not allow multiplexing within the same reaction Alternatively the FlexMAP detection system Luminex
13. the user through the steps required to start image acquisition and data processing 2 Apply optical oil to the undersurface of the sample cartridge This step is important for achieving high quality images The optical oil is applied auto matically in a stand alone unit the nCounter Imaging Oil Applicator that accompanies the Digital Analyzer 3 Open the door located at the top of the analyzer Using a lens paper wipe the objective clean prior to starting image acquisition Place a drop of optical oil on the lens 4 Place the cartridge in the correct orientation into one of the six stage positions Be sure that the cartridge is seated flat in the slot and close the magnetic clips gently The analyzer has six stage positions each of which can be loaded with a cartridge 5 Upload the reporter library file RLF specific to each CodeSet onto the analyzer Save the RLF on the local analyzer drive Each CodeSet is accompanied by a unique RLF which is generated by NanoString and supplied on a memory stick The RLF contains information e g gene names gene symbols accession numbers used during image processing to assign a target identity to each individual barcode This step is performed only once for each user defined CodeSet 6 Create a cartridge definition file CDF and upload onto the local analyzer drive using the memory stick Current Protocols in Molecular Biology BASIC PROTOCOL 2 Discovery of Differentially E
14. A gene expression signature as a predictor of survival in breast cancer N Engl J Med 347 1999 2009 Internet Resources http www nanostring com NanoString Technologies website for detailed infor mation regarding products related literature and applications http www broadinstitute org gsea msigdb index jsp The Molecular Signatures Database MSigDB is a collection of gene sets including positional gene sets curated gene sets motif gene sets computa tional gene sets and gene ontology gene sets https www oncomine org resource login html A database of cancer gene expression profiles http www luminexcorp com Supports a _ bead based_ platform for high throughput gene expression signature analysis for the measurement of up to 100 transcripts in many thousands of samples Discovery of Differentially Expressed Genes 25B 10 17 Supplement 94
15. BASIC HYBRIDIZATION OF TARGET mRNA TO GENE SPECIFIC PROBE PAIRS eee This protocol outlines the hybridization and post hybridization procedures for measuring mRNA levels using the nCounter Gene Expression Assay Hybridization is performed in tube strips that are later loaded into a NanoString Sample Cartridge for post hybridization processing One cartridge can hold up to 12 different reactions samples so the user can set up 1 to 12 hybridization reactions at a time keeping in mind that the cartridge must be discarded after use and cannot be re used Each sample should be hybridized in triplicate The hybridization protocol should be followed exactly as described as the order of addition of components is important When setting up the hybridization reactions it is important not to mix reagents by vortexing or pipetting vigorously as this may shear the reporter probes Mixing should be done by gently flicking or inverting the tubes two to three times If a microcentrifuge is used to collect the reaction mixture at the bottom of the tubes do not spin at more than 1 000 rpm for more than 30 sec and do not pulse spin as this will cause the centrifuge to go to maximum speed which may spin the CodeSet out of solution After hybridization the nCounter Prep Station processes samples to prepare them for sub sequent imaging and data processing Post hybridization processing includes removal of unbound reporter and capture probes attachment of mRNA
16. Digital Multiplexed Gene Expression UNIT 25B 10 Analysis Using the NanoString nCounter System Meghana M Kulkarni Harvard Medical School Boston Massachusetts ABSTRACT This unit presents the protocol for the NanoString nCounter Gene Expression Assay a robust and highly reproducible method for detecting the expression of up to 800 genes in a single reaction with high sensitivity and linearity across a broad range of expression levels The methodology serves to bridge the gap between genome wide microarrays and targeted real time quantitative PCR expression profiling The nCounter assay is based on direct digital detection of mRNA molecules of interest using target specific color coded probe pairs It does not require the conversion of mRNA to cDNA by reverse transcription or the amplification of the resulting cDNA by PCR Each target gene of interest is detected using a pair of reporter and capture probes carrying 35 to 50 base target specific sequences In addition each reporter probe carries a unique color code at the 5 end that enables the molecular barcoding of the genes of interest while the capture probes all carry a biotin label at the 3 end that provides a molecular handle for attachment of target genes to facilitate downstream digital detection After solution phase hybridization between target mRNA and reporter capture probe pairs excess probes are removed and the probe target complexes are aligned and immobilized in th
17. an example of RCC data files and data analysis see the supplementary file at http www currentprotocols com protocol mb25B 10 COMMENTARY Background Information The development of gene expression signa tures that can serve as phenotypes and there fore experimental read outs for various bio logical states represents a powerful tool for the discovery of functional connections between genes diseases and drugs The underlying assumption is that common gene expression changes reflect functional connectivity which is expected to reflect a role in regulating com mon biological processes Lamb et al 2003 Amit et al 2009 Despite the promise and early success of the approach in distinguish ing different cancer types and predicting pa tient survival and response to therapy van de Vijver et al 2002 Glas et al 2005 Burczyn ski et al 2006 the robustness of the approach has not yet been evaluated and validated on a large scale The challenge for the future will be to take these analyses to a higher level by performing high throughput small molecule and RNA interference screens where measur ing the entire transcriptome is neither nec essary nor economically viable An ideal method would provide a direct measurement of each target mRNA of interest using a small amount of starting material and without re quiring cDNA synthesis or amplification Im portantly the method should be amenable to high throughput screening wi
18. ature gene list is selected A Design Report containing the scores for each hybridization parameter for the best reporter capture probe pairs including target sequence recognized by the probe pair is sent to the user as an Excel file for final review 3 The user reviews the Design Report It is especially critical to cross check that the sequence being recognized by the selected probe pairs corresponds to the sequence of the target genes selected by the user in the signature gene list 4 NanoString completes the design and manufacture of the reporter and capture probe pairs approved by the user The entire process from submission of the list of signature genes to design and manufacture of the CodeSet containing reporter capture probe pairs 1s completed in 8 to 10 weeks Probe Replication and Control Probes In any hybridization based assay it 1s critical to design several controls that can be used to determine whether the observed hybridization is specific to the target sequence One approach is to use an alternate sequence for example the mismatch probes on an Affymetrix gene chip or sense probes rather than anti sense to distinguish between specific and non specific or off target hybridization Another commonly used control is to design and use multiple probes for the same gene transcript Given that most gene expression signatures are comprised of tens to several hundred genes and that this approach makes it possible to multiplex up to
19. benchtop centrifuge with a swinging bucket rotor Slide open the door of the Prep Station and empty the solid waste containing tips and liquid waste receptacles Briefly spin down the hybridized samples using a picofuge Remove caps and place the hybridized sample tubes on the deck of the PrepStation ensuring that tube 1 aligns with position 1 The numbering on the deck of the Prep Station corresponds to the numbers on the hybridized sample tubes If the sample tubes are inverted all sample specific information will be assigned to incorrect data files On the Sample Selection touch screen select all wells that are to be processed Remove the clear plastic lids from the Prep Plates and place the plates on the deck of the Prep Station as indicated on the touch screen oriented so the labels face the user Do not remove the foil seal or pierce the wells on the Prep Plates The Prep Station pierces the wells during processing Remove the metal tip carrier from the Prep Station deck by lifting straight up Place the tips and foil piercers into the carrier Replace the loaded tip carrier back onto the Prep Station deck so the foil piercers are closest to the user Place the tip sheaths on the deck and press firmly into place Place two strips of empty tubes without caps on the deck Securely close the lid that flips down over the tubes Open the cartridge pouch and carefully place the cartridge under the electrode fixture in the c
20. bridization reactions are removed from the thermocycler proceed imme diately to post hybridization processing on the nCounter Prep Station Do not store hybridization reactions Set up and run Prep Station 11 Turn on the nCounter Prep Station using the power switch located on the side of the machine The nCounter Prep Station has a user friendly touch screen with which the user can interface with the robot Once powered the Prep Station touch screen guides the user through the steps that must be followed to load the deck and start a run 12 Bring a sealed Sample Cartridge and two Prep Plates to room temperature for at least 10 min before use One Sample Cartridge and two Prep Plates are used to process I to 12 reactions in a single run Prep Plates are 96 well plates containing all reagents for post hybridization processing including magnetic beads for removing unhybridized probe wash buffer and immobilization and imaging reagents Current Protocols in Molecular Biology Discovery of Differentially Expressed Genes 25B 10 9 Supplement 94 13 14 15 16 17 18 19 20 21 22 ZS 24 29 The NanoString nCounter System 25B 10 10 Supplement 94 To prevent accumulation of condensation on the cartridge do not open the pouch seal until it has reached room temperature Collect contents of the Prep Plates in the bottom of the wells by centrifuging for 2 min at 670 x gina standard
21. c 100 base regions are arbitrarily labeled POS_A to POS_G and are generated from the following non human sequences NCBI RefSeq accession number AY058658 1 A E F accession number AY058560 1 B D accession number DQ412624 G In addition the CodeSet includes eight negative control probe pairs arbitrarily labeled NEG_A to NEG_H for which there are no corresponding target sequences These negative control sequences are part of a National Institute of Standards and Technologies NIST effort to generate a set of sequences that can be used to standardize across different gene expression assays including microarrays and PCR based platforms These sequences generated by the External RNA Control Consortium ERCC as part of the NIST are alien in nature 1 e they are synthetic or are from very obscure bacterial species and have been screened against the non redundant sequence database for uniqueness They should yield negative results in all common species used in research labs Devonshire et al 2010 The negative controls are used to calculate background hybridization signal and to determine present absent calls for target genes Sample Preparation A crucial component in a successful nCounter gene expression assay is the generation of high quality total RNA Total RNA from cells and tissues is generally extracted and purified using TRIzol Reagent Invitrogen according to the manufacturer s protocol TRIzol extracted total RNA sho
22. c perturbations on a global scale via high throughput small molecule or RNA interference screens to assess the extent to which these perturbations exhibit the same or distinct molecular phenotypes Lamb et al 2006 Such screening on a global scale using microarrays or RNA Seg can be both technically challenging and prohibitively expensive Current Protocols in Molecular Biology Discovery of Differentially Expressed Genes 25B 10 5 Supplement 94 The NanoString nCounter System 25B 10 6 Supplement 94 Alternatively gene sets that serve as surrogates for a biological state can be as sembled from public databases A number of public databases provide excellent re sources for building useful gene expression signatures including MSigDB developed at the Massachusetts Institute of Technology and the Broad Institute Subramanian et al 2005 and the Oncomine cancer profiling database developed at the Univer sity of Michigan Ann Arbor Rhodes et al 2004 2005 Rhodes and Chinnatiyan 2005 NanoString also offers pre built gene expression panels and Virtual Gene sets http www nanostring com products assays that are composed of genes to study sig naling pathways disease states and a number of other biological processes in human and mouse systems While assembling a set of genes to represent the gene expression signature of a state of interest it is important to select genes whose expression levels span a broad range of
23. d on present absent calls The R value of the linear fit to this data is 0 9999 B Scatter plot of nor malized background subtracted counts for all 100 genes assayed shown in log scale for technical replicates of insulin stimulated S2R samples S2R _INS40_1 vs S2R _INS40_2 Genes were not filtered based on detection The F value of a linear fit to this data is 0 9998 The normalized background subtracted counts used to construct both graphs are available in the supplementary file at http www currentprotocols com protocol mb25B 10 Current Protocols in Molecular Biology Discovery of Differentially Expressed Genes 25B 10 15 Supplement 94 The NanoString nCounter System 25B 10 16 Supplement 94 Time Considerations The entire protocol from hybridization to data collection and processing requires 2 days but hands on time is less than 30 min It takes 5 10 min to set up 12 hybridization re actions excluding the time required to thaw reagents and the hybridization reaction pro ceeds overnight for 12 16 hr The set up for the Prep Station takes 10 min and post hybridization processing of 12 samples re quires lt 2 5 hr The Digital Analyzer takes 4 5 hr to process 12 samples at a 600 FOV resolution and can analyze 72 samples per day running unattended in continuous mode In this case the hybridizations should be stag gered so that when post hybridization process ing of one cartridge is completed on the Prep
24. e nCounter cartridge which is then placed in a digital analyzer for image acquisition and data processing Hundreds of thousands of color codes designating mRNA targets of interest are directly imaged on the surface of the cartridge The expression level of a gene is measured by counting the number of times the color coded barcode for that gene is detected and the barcode counts are then tabulated Curr Protoc Mol Biol 94 25B 10 1 25B 10 17 2011 by John Wiley amp Sons Inc Keywords gene expression signature e multiplex analysis e signal transduction e high throughput screening e molecular barcode INTRODUCTION Virtually any biological condition whether a developmental state a cellular response to extracellular stimulus or a pathological state is reflected by changes in gene expression One measure of the expression level of a gene is the number of corresponding mRNA molecules present in the cell While the expression level of a single gene cannot define biological states the ability to measure gene expression on a genome wide scale using technologies such as microarrays provides an opportunity to identify gene expression signatures relevant to a particular biological phenomenon or response Gene expression signatures are commonly composed of tens to hundreds of genes Amit et al 2009 The collective expression pattern of genes within a particular signature has been used in a number of studies to predict disease type and progressi
25. eat the thermocycler to 65 C Program the thermocycler using a 30 ul volume calculated temperature mode heated lid and forever time setting Do not set the thermocycler to ramp down to 4 C at the end of the run Alternatively a 65 C hybridization oven may be used with a large beaker full of water placed in the oven to ensure a humid environment It is ideal to maintain a constant temperature of 65 C during hybridization to ensure optimal reaction conditions Avoid using a water bath or heat block since evaporation over time could cause fluctuations in temperature 8 Gently flick the tubes containing capture probe and briefly spin down contents Add 5 ul capture probe set to each tube and immediately place tubes at 65 C Cap tubes and mix the reagents by gently flicking the tube strips several times Briefly spin down contents and immediately place the tube strip in the 65 C thermocycler Minimizing the time between adding the capture probe and placing the reaction at 65 C will increase the sensitivity of the assay The final hybridization reaction for each total RNA sample contains 5 ul sample RNA 150 ng 10 wl reporter probe final 40 pM 5 ul capture probe final 200 pM and 10 ul hybridization buffer 5x SPPE pH 7 5 with 0 1 Tween 20 9 Incubate hybridization reactions at 65 C for at least 12 hr optimally 16 hr Maintain reactions at 65 C until ready for processing but do not exceed 30 hr 10 Once the hy
26. erent excitation wavelengths 480 545 580 and 622 nm corresponding to the four different fluorescent dyes in the barcode At the highest resolution images of each field of view are acquired at 60x magnification 600 fields of view to yield hundreds of thousands of counts of target mRNA molecules The acquired digital images are then processed internally on the analyzer The expression level of a gene is measured by counting the number of times its specific barcode is detected and the barcode counts are tabulated and reported in a CSV format In this system the number of reporter molecules counted determines the number of images acquired This feature of the analyzer contributes in part to the large dynamic range and high sensitivity of the assay This protocol briefly describes the steps involved in image acquisition For a more detailed step by step description of the process refer to the nCounter Digital Analyzer User Manual provided at http www nanostring com Materials nCounter Digital Analyzer NanoString Technologies no NCT DIGA 120 nCounter Imaging Oil Applicator and optical oil provided with Digital Analyzer nCounter Memory Stick NanoString Technologies provided with CodeSet Personal computer with Microsoft Excel Collect data 1 Turn on the Digital Analyzer using the power switch located at the back of the machine Similar to the Prep Station the Digital Analyzer has a user friendly touch screen interface that guides
27. is approach depends on the ability to screen a large number of genes and compounds in a timely and cost effective manner Therefore quantitative procedures for high throughput measurement of mRNA levels in a simple flexible and cost effective manner are important This unit presents the protocol for the nCounter Gene Expression Assay http NanoString com a robust and highly reproducible method for detecting the expression of up to 800 genes in a single reaction The methodology serves to bridge the gap between genome wide microarrays and targeted real time quantitative PCR expression profiling by virtue of its ability to multiplex up to 800 gene transcripts within a single reaction with high sensitivity and linearity across a broad range of expression levels The nCounter assay is based on direct imaging of mRNA molecules of interest that are detected using target specific color coded probe pairs Geiss et al 2008 It does not require the conversion of mRNA to cDNA via reverse transcription or the amplification of the resulting cDNA via PCR The large dynamic range of the assay and the lack of any enzymatic reactions allows direct analysis of a wide variety of sample types including purified total RNA crude cell and tissue lysates RNA extracted from formalin fixed paraffin embedded FFPE tissues and blood collected without globin mitigation Each target mRNA of interest is detected using a pair of probes called the reporter and capt
28. is of gene expression Eur J Neurosci 16 409 413 Geiss G K Bumgarner R E Birditt B Dahl T Dowidar N Dunaway D L Fell H P Ferree S George R D Grogan T James J J Maysuria M Mitton J D Oliveri P Osborn J L Peng T Ratcliffe A L Webster P J Davidson E H Hood L and Dimitrov K 2008 Direct multiplexed measurement of gene expression with color coded probe pairs Nat Biotechnol 26 317 325 Glas A M Kersten M J Delahaye L J Witteveen A T Kibbelaar R E Velds A Wessels L F Joosten P Kerkhoven R M Bernards R van Krieken J H Kluin P M van t Veer L J and de Jong D 2005 Gene expression profiling in follicular lymphoma to assess clinical aggressiveness and to guide the choice of treatment Blood 105 301 307 Golden T R Hubbard A Dando C Herren M A and Melov S 2008 Age related be haviors have distinct transcriptional profiles in Caenorhabditis elegans Aging Cell 17 850 865 Hsuih T C Park Y N Zaretsky C Wu F Tyagi S Kramer F R Sperling R and Zhang D Y 1996 Novel ligation dependent PCR assay for detection of hepatitis C in serum J Clin Micro biol 34 501 507 Lamb J Ramaswamy S Ford H L Contreras B Martinez R V Kittrell F S Zahnow C A Patterson N Golub T R and Ewen M E 2003 A mechanism of cyclin D1 action encoded in the patterns of gene expression in human can cer
29. is shown on the right D The loaded sample cartridge is transferred to the Digital Analyzer left for imaging and data processing A raw image of color coded reporter probes bound to complementary target mRNA is shown middle along with a schematic of the tabulated counts for target genes right Modified with permission from NanoString Technologies 25B 10 4 Supplement 94 Current Protocols in Molecular Biology common sequence of the capture probe washed in a buffer containing low salt and eluted off the beads at elevated temperature This step removes excess unbound reporter probes partial complexes containing only the reporter and target mRNA and non target cellular transcripts not bound to a capture probe The eluate containing capture probes and tripartite complexes is then purified with a second round of beads with the common sequence of the reporter probe to remove excess unbound capture probes Second the purified complexes are loaded into the NanoString Sample Cartridge using hydrostatic pressure and bound to the streptavidin coated surface via the 3 biotin label of the capture probe Each cartridge contains 30 um deep microfluidic channels that can be loaded with twelve different samples at a time After attachment to the surface of the cartridge the bound molecules are extended and aligned by applying an electric field 160 V cm for 1 min along each of the twelve flow cells The extended complexes are further immobilized a
30. led to a series of six complementary RNA segments each labeled with one of four spectrally non overlapping fluorescent dyes The linear arrangement of the differently colored RNA segments creates a unique color code for each target gene of interest The expression level of each gene is determined by scoring the number of times its corresponding color code is detected In addition to the target specific probe pairs the CodeSet contains unique pairs of capture and reporter probes for reference genes specified by the user as well as a number of nCounter sys tem controls including eight negative controls and seven positive spike in controls for which RNA targets are added to the reactions The methodology offers the flexibility of multiplexing up to 800 reporter capture probe pairs within a single reaction The CodeSet is mixed in large excess with the mRNA sample in a solution phase hy bridization reaction Fig 25B 10 1B Hybridization results in the formation of tripartite complexes each containing a target mRNA bound to its specific capture and reporter probes Each total RNA sample is hybridized in triplicate with each reaction containing 150 ng total RNA 200 pM of each capture probe and 40 pM of each reporter probe The hybridization reaction is carried out in a standard thermocycler for 16 hr Post hybridization processing is carried out on a fully automated nCounter Prep Station liquid handling robot Fig 25B 10 1C First unhybridized p
31. on e g van de Vijver et al 2002 Burczynski et al 2006 More importantly gene signature based analysis has the potential to define the molecular mechanism s by which genes contribute to a particular biological process and can thus not only inform the choice of therapeutic intervention but also predict patient response to therapy Lamb et al 2003 2006 Glas et al 2005 Discovery of Differentially Expressed Genes Current Protocols in Molecular Biology 25B 10 1 25B 10 17 April 2011 25B 10 1 Published online April 2011 in Wiley Online Library wileyonlinelibrary com DOI 10 1002 0471142727 mb25b10s94 Supplement 94 Copyright 2011 John Wiley amp Sons Inc The NanoString nCounter System 25B 10 2 Supplement 94 The ability to identify specific gene expression signatures that reflect different bio logical states raises the possibility of performing high throughput small molecule or RNA interference screens using a signature of interest as the read out Such screening would allow interrogation of pharmacological and genetic perturbations to assess the extent to which they exhibit the same or distinct signatures Lamb et al 2006 Link ing biological states with specific perturbations genetic and or pharmacological using quantitative information from changes in the expression of corresponding signature genes can provide a global understanding of the organization of cellular signaling circuits The success of th
32. orrect orientation Make sure that it is seated completely in the machined depression Carefully lower the electrode fixture in place over the cartridge so the 24 electrodes insert into the 24 wells If any resistance is felt while lowering the fixture stop and make sure that the electrodes are correctly aligned Close the door of the Prep Station by pulling down Press Start on the touch screen to begin processing The Prep Station first checks that all consumables and reagents have been placed in the correct orientation To do this the Prep Station confirms that the sensors for the sample cartridge electrode fixture and heater lid are all in the correct state The pipet head then checks that tips tip sheaths strip tubes and Prep Plates are all in place by touching them with a set of validation tips Upon completion of the run remove the cartridge from the deck taking care not to bend the electrodes Seal the cartridge using cartridge well seals and label with a date and unique identifier Target mRNA molecules with unique barcodes are now ready to be imaged counted and scored for relative abundance measurements Current Protocols in Molecular Biology DATA COLLECTION AND ANALYSIS The nCounter Digital Analyzer acquires images of the immobilized fluorescently labeled target mRNA molecules in the sample cartridge using a CCD camera and a microscope objective lens The analyzer takes images of each field of view FOV using four diff
33. oting There are three important parameters in determining the success of this digital mul tiplexed gene expression assay First and foremost is the development of a useful gene expression signature that reflects the biological state of interest For details on pattern matching algorithms that are com monly employed to develop robust gene ex pression signatures from publicly available S2 1 E S2 2 Xx S2 3 X S2R _INS40_1 X S2R _INS40_2 S2R _INS40_3 Avg S2R E Avg S2R _INS40 Linear Avg S2R Linear Avg S2R _INS40 Figure 25B 10 2 Linearity and reproducibility of the positive spike in controls DNA oligonucleotide targets were spiked into each sample at concentrations of 0 1 0 5 1 5 10 50 and 100 fM No target was added for the negative control probe pairs A Signal counts on a log scale vs concentration of the spike on a log scale Each of three replicate measurements for each spike in baseline Drosophila S2R cells S2R _1 2 3 and insulin stimulated S2R cells S2R _INS40_1 2 3 is shown The replicate measurements overlap completely except at the lowest concentrations B Average signal vs concentration on a linear scale for positive spike in controls in both baseline and insulin stimulated Drosophila S2R samples The correlation coefficients FR of the linear fit to the average signal are 0 9786 and 0 9803 for baseline and insulin stimulated samples respectively The normalized background
34. reporter capture complexes to streptavidin coated Sample Cartridges and extension and alignment of the complexes by application of an electric field The Prep Station can process up to 12 samples per run in approximately 2 hr For a general description of the post hybridization process see Strategic Planning For a detailed description of the Prep Station including step by step images maintenance and troubleshooting information refer to the nCounter Prep Station User Manual provided at http www nanostring com Materials Sample see Strategic Planning total RNA 150 ng per hybridization reaction 80 C or cell lysate from 2 500 10 000 cells 80 C nCounter GX CodeSet probe pairs for user defined target genes NanoString Technologies 80 C Hybridization buffer room temperature provided in nCounter Prep Pack RNase free DNase free water nCounter Prep Pack NanoString Technologies including racked tips and foil piercers 12 tube strips and caps tip sheaths for storing tips to prevent cross contamination and unnecessary tip consumption and cartridge well seals store at room temperature Tube Strip PicoFuge Stratagene Thermocycler or hybridization oven nCounter Prep Station NanoString Technologies no NCT PREP 120 nCounter Sample Cartridges NanoString Technologies store at 20 C nCounter Prep Plates foil sealed 96 well plates NanoString Technologies store at 4 C Benchtop centrifuge with
35. robes are sequentially re moved using magnetic beads coupled to short oligonucleotides complementary to the common sequence tags on the capture and reporter probes orange and black segments in Fig 25B 10 1A The hybridization reaction is first purified using beads with the Current Protocols in Molecular Biology Discovery of Differentially Expressed Genes 25B 10 3 Supplement 94 A 3 biotin 5 color code signal target specific capture probe target specific reporter probe sample total RNA lysate codes are counted and tabulated Figure 25B 10 1 Overview of nCounter Gene Expression Assay A Schematic of target specific capture and reporter probes showing the 5 reporter barcode and the 3 biotin capture handle The orange segment in the capture probe represents a 30 base sequence common to all capture probes and the black segment in the reporter probe represents a 60 base sequence common to all reporter probes Both are used to remove excess unbound probe B During hybridization buffer the CodeSet capture and reporter probe library and sample total RNA or cell lysate are combined in strip tubes and hybridized overnight at 65 C Hybridization results in the formation of tripartite structures comprising target MRNA bound to specific capture and reporter probes C Strip tubes containing tripartite complexes are placed on the Prep Station left for automated post hybridization processing The deck layout
36. subtracted counts used to construct both graphs are available in the supplementary file at htto vww currentprotocols com protocol mb25B 10 25B 10 14 Supplement 94 Current Protocols in Molecular Biology microarray data refer to the review by Nevins and Potti 2007 and the article by Amit et al 2009 Also refer to public databases such as the Molecular Signatures Database MSigDB and Oncomine for collections of available gene sets The second important parameter is the use of multiple independent probe pairs for each mRNA target of interest and a number of dif ferent reference genes that show constant gene expression levels across experiments Third as with any gene expression assay the quality of the total RNA preparation must be veri fied before proceeding with the hybridization and downstream processing Results using cell lysate samples are highly correlated with those from total RNA gt 0 95 for both low and high expressing genes however this is only possi ble if the cell lysate is used immediately after preparation without freeze thaw cycles Since this is an enzyme free system 1 e no PCR or reverse transcription it is likely that cell lysate counts correlate better with total RNA in this system than in others It is always preferable to have at least three probe pairs per target gene It is also highly gt Normalized counts S2Rt _2 recommended to include multiple eight to ten reference genes since
37. swinging bucket rotor for plates e g Eppendorf NOTE Sample Cartridges Prep Plates and Prep Packs can be purchased together in a comprehensive nCounter Master Kit Perform hybridization The NanoString 1 Remove cell lysate or total RNA samples and aliquots of reporter and capture probe nCounter System reagents from the 80 C freezer and thaw on ice for 30 min Invert several times to mix well and briefly spin down contents of the tubes 25B 10 8 Supplement 94 Current Protocols in Molecular Biology 2 Prepare a master mix containing 130 ul reporter probe and 130 ul hybridization buffer Mix by gently flicking the tube and briefly spin down contents 3 Dilute total RNA or cell lysate samples with RNase free DNase free water to give a total RNA concentration of 30 ng ul For lysates 10 000 mammalian cells is the equivalent of 100 ng total RNA 4 Label a 12 tube strip so that the sample in each tube is clearly indicated Use triplicate tubes for each sample Cut the tube strips in half to fit in a picofuge 5 Add 20 ul master mix to each of the 12 labeled strip tubes It is advisable to use a fresh tip for each pipetting step to accurately pipet the correct volume 6 Add 5 ul diluted total RNA or cell lysate sample containing 100 150 ng total RNA to appropriately labeled triplicate strip tubes containing master mix Mix by gently flicking the tubes and briefly spin down contents using the tube strip picofuge 7 Pre h
38. t the reporter probes by the addition of biotinylated oligonucleotides complementary to the 5 end of each reporter probe After immobilization a SlowFade reagent is added to each flow cell prior to imaging The NanoString cartridges containing the immobilized complexes are sealed and imaged on the nCounter Digital Analyzer Fig 25B 10 1D which takes images of each field of view FOV using four different excitation wavelengths 480 545 580 and 622 nm cor responding to the four fluorescent dyes At the highest resolution images of each field of view are acquired at 60x magnification 600 fields of view using a microscope objective and a CCD camera yielding hundreds of thousands of counts of target mRNA molecules The acquired digital images are processed on the Digital Analyzer The expression level of a gene 1s measured by counting the number of times the specific barcode is detected and the barcode counts are tabulated in a comma separated value CSV format Selection of Gene Signature In order to use changes in gene expression to link genes drugs and or diseases to specific biological processes it is imperative to establish useful expression signatures that robustly reflect the specific biological states of interest An expression signature which serves as a surrogate for a biological state of interest can be defined as a set of genes whose pattern of expression is characteristic of that state Thus the first step is to define a
39. th sensitivity that is higher than that of microarrays and about equal to that of TaqMan RT PCR NanoString s nCounter Gene Expression Assay is a novel technology based on direct measurement of target MRNA molecules The technology uses color coded molecular bar codes to designate each individual mRNA of interest and single molecule imaging to de tect and count up to 800 unique transcripts in a single reaction The single molecule imaging provides a large dynamic range and the assay as a whole offers significantly higher levels Current Protocols in Molecular Biology of precision and sensitivity compared to mi croarray gene expression profiling Compared to surface bound hybridization in the case of microarrays the solution phase hybridization of this method offers low background signals significantly increasing the detection of low abundance mRNAs Measured expression lev els show consistently tight coefficients of vari ation CV across a broad range of relative mRNA abundance levels ranging from lt 20 all the way down to a sensitivity of a single copy per cell One of the biggest advantages of the assay is that it does not require the con version of mRNA to cDNA or amplification of the resulting cDNA The assay can be cus tomized to measure target genes defined by the user and offers true multiplexing capability of up to 800 genes in a single reaction Unlike real time quantitative PCR a single reaction is required per
40. the counts for each target gene and the control genes by the normalization factor for that sample flow cell Calculate the average normalized background count for a sample flow cell from the average of the eight negative control counts Subtract this value from the normalized count of each gene target in that sample flow cell to yield normalized background subtracted counts for each target gene To determine if the normalized background subtracted counts are statistically above background perform a Student s test against the eight human negative controls A gene is considered to be above background if the average count for the target gene is greater than the average counts for the eight negative control genes and if the P value of the t test is less than 0 05 Current Protocols in Molecular Biology The t test assumes that the values being compared follow a normal distribution and that the variances in the data are equal in the two groups being compared If the data are not normally distributed the Mann Whitney U test also called the Mann Whitney Wilcoxon MWW or Wilcoxon rank sum test can be used or Welch s t test can be used if the variances between the two groups being compared are highly unequal 16 Average triplicates for each sample 17 Calculate relative changes in the abundance of target mRNA molecules using the normalized background subtracted counts for one or more reference genes included in the CodeSet For
41. uld be further purified using RNeasy mini spin columns Qiagen according to the manufacturer s protocol The concentration and quality of the total RNA preparation should determined by measuring absorbance at 260 and 280 nm us ing a standard UV spectrophotometer or the Nanodrop system Thermo Scientific Each hybridization reaction requires 100 150 ng of total RNA The integrity of the total RNA preparation should be verified by denaturing PAGE or on a Bioanalyzer model 2100 Agilent Technologies before proceeding with the hybridization reaction RNA should be aliquoted to avoid freeze thaw cycles and stored for up to several years at 80 C Alternatively hybridization can be set up using cell lysates Cell lysates should be prepared using an RNeasy mini kit Qiagen according to manufacturer s recommen dations but using a concentration of 2 500 to 10 000 cells ul of RLT buffer Qiagen The nCounter cell lysate hybridization protocol is optimized for 10 000 mammalian cells reaction which is the equivalent of 100 ng total RNA Lysates should be used immediately after preparation to maintain the integrity of the cellular RNA For more detailed descriptions of the manufacturer s Total RNA Standard Proto col and Cell Lysate Protocol see the nCounter Gene Expression Assay Manual at http www nanostring com Current Protocols in Molecular Biology Discovery of Differentially Expressed Genes 25B 10 7 Supplement 94
42. ure probes Each probe within a probe pair contains target specific sequences that to gether recognize a 100 base contiguous region within the targeted mRNA Fig 25B 10 1 In addition the reporter probes carry a color code at the 5 end that enables the molecular barcoding of the genes of interest while the capture probes carry a biotin label at the 3 end that provides a molecular handle for the attachment of target genes to facilitate down stream digital detection The color code on each reporter probe has six positions each of which can be one of four spectrally non overlapping fluorescent colors The different combinations of the four distinct colors at six contiguous positions allows for a large diversity of color based barcodes each designating a different gene transcript that can be mixed together in a single reaction for hybridization and still be individually resolved and identified It is theoretically possible to generate 4 4 096 different color codes each barcoding a distinct mRNA However the kinetics of solution phase hybridization and the technology for digital detection of different color barcodes currently limit the measurement power to less than a thousand mRNA species The target mRNA is mixed in solution with a large excess of the reporter and capture probe pairs so that each targeted transcript finds its corresponding probe pair After hybridization excess unbound probes are washed away and the tripartite complexes
43. xpressed Genes 25B 10 11 Supplement 94 The NanoString nCounter System 25B 10 12 Supplement 94 11 A CDF is created by the user for every cartridge analyzed The CDF defines sample specific data to associate with the data output It also defines parameters used by the analyzer during image collection and processing such as Lane ID for each of the twelve flow cells in the cartridge Sample ID the name of the sample contained in each lane or flow cell GeneRLF the CodeSet RLF to associate with the data and FOVcount number of images to analyze per assay flow cell At the highest resolution the FOVcount is set to 600 FOVs A CDF template is included on the memory stick and can be opened in Excel on the user s personal computer to create a CDF file for each cartridge Select Start Counting from the Main Menu on the touch screen Select the cartridge position corresponding to the stage where the sample cartridge has been loaded Select the cartridge specific CDF that was saved to the local analyzer drive 10 Shut the door of the instrument and select Start to begin image acquisition and data processing The data are collected in a reporter code count RCC file created for each flow cell in the cartridge Each RCC file contains the number of counts for each target mRNA molecule for one flow cell corresponding to one sample A separate RCC file is created for each flow cell or sample in the cartridge
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