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PrimeFlow™ RNA Assay Technology Validation Paper Introduction PrimeFlow™ RNA Assay reveals the dynamics of RNA and protein expression within individual cells, facilitating unprecedented analysis of their correlation as the cells change over time or in response to stimulation. This novel assay uses fluorescent in situ hybridization (FISH) to enable simultaneous detection of as many as three RNA transcripts in a single cell using a standard flow cytometer. PrimeFlow RNA Assay is compatible with cell surface and intracellular staining, using common flow cytometry fluorochromes. The assay is based upon proven and well-published ViewRNA assays designed for microscopic analysis of RNA in cells and tissues that combine paired oligonucleotide probe design with branched DNA (bDNA) signal amplification to robustly detect gene expression at the single-cell level. Coupling RNA expression with protein detection on a flow cytometer generates multiparametric data in heterogeneous cell populations and offers in-depth and high content details at the single-cell level. In contrast, microarrays and sequencing can provide comprehensive gene expression data in bulk sample preparations; however, the analysis of bulk samples can mask the individual effects of unique cellular subsets. Using PrimeFlow RNA Assay, specific cell populations may be analyzed for unique transcript expression levels, or cell subsets evaluated over time to determine transcriptional regulation and protein expression simultaneously. Such unique and valuable insights are highly applicable to answering previously unanswerable questions and have broad implications for advancing research across multiple fields of biology. n n n n n Observe the heterogeneity of gene expression at a single-cell level Correlate RNA and protein kinetics within the same cell Detect non-coding RNA in cell subsets Evaluate viral RNA expression in infected cells Analyze mRNA expression levels when antibody is unavailable Figure 1: Example data set Ki-67 RNA vs CD8 Granzyme B Protein vs CD8 Protein vs RNA RNA vs CD8 Protein vs CD8 Protein vs RNA CD8 PE-eFluor® 610 CD8 PE-eFluor® 610 Ki-67 mRNA Alexa Fluor® 488 CD8 PE-eFluor® 610 GrzB Protein PE-Cyanine7 GrzB Protein PE-Cyanine7 GrzB mRNA Alexa Fluor® 750 Ki-67 Protein eFluor® 450 Ki-67 Protein eFluor® 450 Ki-67 mRNA Alexa Fluor® 488 Unstimulated: CD8 PE-eFluor® 610 GrzB mRNA Alexa Fluor® 750 CD8 PE-eFluor® 610 Ki-67 mRNA Alexa Fluor® 488 GrzB Protein PE-Cyanine7 GrzB Protein PE-Cyanine7 GrzB mRNA Alexa Fluor® 750 CD8 PE-eFluor® 610 Ki-67 Protein eFluor® 450 Ki-67 Protein eFluor® 450 Ki-67 mRNA Alexa Fluor® 488 2-day Anti-CD3/CD28 stimulated: CD8 PE-eFluor® 610 CD8 PE-eFluor® 610 GrzB mRNA Alexa Fluor® 750 PrimeFlow™ RNA Assay in action C57Bl/6 splenocytes were unstimulated (top row) or stimulated for 2 days with Anti-Mouse CD3 and CD28 Functional Grade Purified antibodies (cat. no. 16-0031 and cat. no. 16-0281) (bottom row) and in the presence of Protein Transport Inhibitor Cocktail (cat. no. 00-4980) for the last 3 hours of culture, followed by analysis using the PrimeFlow™ RNA Assay (cat. no. 88-18001). Cells were fixed and permeabilized using the PrimeFlow™ RNA Assay buffers and protocol, then intracellularly stained with Anti-Mouse CD8a PE-eFluor ® 610 (cat. no. 61-0081), Anti-Mouse Ki-67 eFluor ® 450 (cat. no. 48-5698), and Anti-Mouse Granzyme B PE-Cyanine7 (cat. no. 25-8898). Cells were then hybridized with Type 6 Mouse Granzyme B Alexa Fluor ® 750 (cat. no. VB6-16522), Type 4 Mouse Ki-67 Alexa Fluor ® 488 (cat. no. VB4-16518), and Type 1 Mouse β-actin Alexa Fluor ® 647 (cat. no. VB1-10350) target probes. Assay technology Fluorescent in situ hybridization (FISH) is a powerful technique that allows specific localization of ribonucleic acid targets in fixed cells. The basic premise of the application relies on detecting nucleic acids through sequential hybridization of nucleic acid probes that provides gene expression information at a single-cell level. Traditional FISH techniques are generally limited by high background and low sensitivity due to non-specific binding and inefficient signal amplification. PrimeFlow™ RNA Assay incorporates a proprietary oligonucleotide probe set design and branched DNA (bDNA) signal amplification technology to analyze RNA transcripts by flow cytometry. bDNA technology provides a unique approach to RNA detection and signal amplification by amplifying the reporter signal rather than the target sequence (e.g., PCR) for consistent results, a common problem for PCR-based assays. Figure 2: Branched DNA (bDNA) probe design principle Label Probe Label Probe Amplifier Amplifier Pre-amplifier Pre-Amplifier In the PrimeFlow™ RNA Assay, target-specific probe sets contain 20 to 40 oligonucleotide pairs that hybridize to the target RNA transcript. Signal amplification is achieved through specific hybridization of adjacent oligonucleotide pairs to bDNA structures, formed by Pre-amplifiers, Amplifiers, and fluorochrome-conjugated Label Probes, resulting in excellent specificity, low background, and high signal-to-noise ratio (Figure 2). RNA RNA Left Oligo RightOligo Oligo Right Figure 3: PrimeFlow™Sample RNA Assay workflow Sample Preparation Sample Sample Preparation Sample Sample Preparation Preparation Sample preparation Preparation Preparation (Antibody staining staining Label(Antibody proteins with (Antibody staining optional) (Antibody staining antibody (optional) optional) (Antibody staining (Antibody staining (Antibody staining optional) optional) Fix and cells Fix permeabilize and Permeabilize optional) optional) Fix and Permeabilize suspension cells in suspension Fix and Permeabilize suspension cells FixFix and Permeabilize and Permeabilize Fix suspension and Permeabilize cells Label intracellular proteins suspension cells suspension cells suspension cells with antibodies (optional) Gene 1 Gene 1 Gene 1 2 Gene Gene 1 Gene Gene Gene 1 12 Gene 2 Gene 2 Gene Gene 2 2 Suspension cells Suspension cells with RNA fixed Suspension cells with RNAcells fixed Suspension Suspension cells Suspension cells with RNA fixed Suspension cells with RNA fixed with RNA fixed with withRNA fixedfixed RNA 2 Target Target Hybridization Target Target Hybridization Target Target Hybridization Hybridization Target hybridization Hybridization Hybridization Gene-specific Label Gene-specific Label Extenders (LE) Gene-specific Label Extenders (LE) Gene-specific Label Gene-specific Gene-specific Label Gene-specific Label Extenders (LE) Extenders (LE) Label Extenders Extenders (LE)(LE) Extenders (LE) Gene-specific Gene-specific Blocking Probes (BL) Gene-specific Blocking Probes (BL) Gene-specific Gene-specific Gene-specific Blocking Probes (BL) Blocking Probes (BL) Blocking Probes (BL) Blocking Probes (BL) Incubate cells with Incubate cells with Incubate cells with gene-specific probe Gene Specific Incubate cells with Gene Specific sets (Type 1, 4, or 6) Incubate cells with probe sets probe sets Incubate cells with Incubate cells Gene Specific probe sets with Gene Specific (Type 1, 4 or 6) Gene Specific Gene Specific probe sets (Type 1, 4 or 6) probe sets probe sets probe sets (Type 1, 4 or (Type 1, 4 or 6) 6) (Type 4 or (Type 1, 41,or 6) 6) Signal Signal Amplification Signal Signal Amplification Signal Signal Amplification Amplification Signal amplification Amplification Amplification PreAmplifier PreAmplifier PreAmplifier PreAmplifier Pre-amplifier PreAmplifier PreAmplifier Amplifier Amplifier Amplifier Amplifier Amplifier Amplifier Amplifier Hybridization of Hybridize with Hybridization of Pre-Amplifier and Hybridization of Pre-amplifier andofand Pre-Amplifier Hybridization Amplifier DNA Hybridization Hybridization of of Pre-Amplifier and Amplifier Amplifier DNADNA Pre-Amplifier and (Type 1, 4, and 6) Pre-Amplifier and Pre-Amplifier and Amplifier (Type 4, and 6) (Type 1, 4, 1, orDNA 6)DNA Amplifier Amplifier DNA Amplifier DNA (Type 1, 4, and 6) (Type 1, 4, and 6) (Type 1, and 4, and (Type 1, 4, 6) 6) Detection Detection Detection Detection Detection Detection Detection Label Fluorescent Fluorescent Label Probe Fluorescent Label Probe Fluorescent Label Fluorescence-labeled probes Fluorescent Label Fluorescent Probe Label Probe Label Probes to Add Add labeled probes to cells Probe Probe Add Label Probes to cells Add Label Probes cells Add Label Probes to to Add Label Probes Add Label Probes to to cells cells cells cells process cells using process cells using a standard Flow process cells using a standard Flow process cells using Process using Cytometer Instrument process cells using process cells using a standard Flow Cytometer Instrument a standard Flow cytometer aflow standard Flow a astandard Flow Cytometer Instrument Cytometer Instrument Cytometer Instrument Cytometer Instrument CD8 mRNA AlexaFluor® 647 Left Oligo CD8 PE-Cyanine7 PrimeFlow™ RNA Assay principle The assay workflow contains several steps: surface antibody staining; fixation and permeabilization; intracellular antibody staining, followed by target probe hybridization, with RNA-specific probe sets; signal amplification using bDNA constructs and detection by flow cytometry. For simplicity, detection of only two RNA targets are shown in orange and yellow (Figure 3, Page 2) with only three of the 20 to 40 oligonucleotide target probe pairs per target RNA. Antibody staining, fixation, and permeabilization Single-cell suspensions can be stained for cell surface markers and fixable viability dyes before the cells are fixed and permeabilized. Subsequently, the cells may be stained with antibody directed to intracellular targets, such as transcription factors and cytokines. Spacers After an additional fixation step, the cells are ready to proceed through the hybridization and signal amplification steps. mRNA Target hybridization Target probes (pairs) A target-specific Probe Set (”Roots”) contains 20 to 40 oligonucleotide pairs that hybridize to specific regions across the target RNA sequence. (minimum pairs per mRNA) Subsequent signal20amplification requires that each half of a given oligonucleotide pair binds to the target RNA in adjacent positions. Three types of Probe Sets are currently available to allow detection of RNA labeled with Alexa Fluor® 647 (Type 1 Probe Sets), Alexa Fluor® 488 (Type 4 Probe Sets), or Alexa Fluor® 750 (Type 6 Probe Sets). When detecting more than one RNA target in a single sample, each Probe Set must be a unique type to differentiate its signal from the others. Figure 4A: Probe Set hybridization mRNA Probe Set Probe Set Type Fluorochrome Label Excitation Wavelength (max) Emission Wavelength (max) Laser Excitation Wavelength Bandpass Filter Recommendation Type 1 Alexa Fluor® 647 647 nm 668 nm 632-640 nm 660/20 Type 4 Alexa Fluor® 488 488 nm 519 nm 488 nm 530/30 Type 6 Alexa Fluor 750 749 nm 775 nm 632-640 nm 780/60 ® Signal amplification Signal amplification using bDNA technology is achieved through a series of sequential hybridization steps, that forms a tree-like structure. Pre-amplifier molecules hybridize to their respective pair of bound oligonucleotide probes to form the trunk of the tree. Label Probes (”Leaves”) (Fluorochrome-conjugated oligo Multiple Amplifier molecules hybridize to their respective Pre-amplifier to create the branches. Finally, multiple Label Probes hybridize Amplifier (”Branches”) Bind to multipe sites on Amp Bind to multiple sites on PreAmp to the Amplifiers and form the “leaves” of the tree. A fully assembled signal amplification tree contains 400 Label Probe binding sites. If all target-specific oligonucleotides in a 20 oligonucleotide pair Probe Set bind to the target RNA transcript, an 8,000-fold amplification can be achieved. Fig 3a Branched DNA Tree Assembly Figure 4B: Pre-amplifier (“trunk”) binds to a Probe Set Figure 4C: Amplifier (“branches”) binds to multiple sites on Pre-amplifier Figure 4D: Label Probes (“leaves”) fluorochrome-conjugated oligo binds to multiple sites on Amplifier Fluorescence detection Upon completion of the assay protocol, target RNA data is detected in cells by analyzing the sample on a standard flow cytometer equipped with 633-647 nm and 488 nm lasers and appropriate filter configurations, to capture the fluorescent signals (see table in Figure 4A). 3 Precision Intra-assay variability To assess intra-assay variability, samples from stimulated and unstimulated human peripheral blood mononuclear cells (PBMC) were divided into seven tubes and assessed for expression of Ribosomal Protein L13a (RPL13a), a positive control gene expressed in all PBMC, and interferon gamma (IFNγ), induced only upon stimulation in a subset of lymphocytes. As shown in Figures 5A and 5B, the assay shows robust intra-assay performance, with a CV% less than 10% for both RPL13a and IFNγ. Figure 5A Figure 5B 6000 6000 10.7 1600 1600 RPL13a Alexa Fluor® 750 MedFl IFNγ Alexa Fluor® 750 MedFl 1800 1800 1400 1400 1200 1200 1000 1000 800 800 600 600 400 400 200 200 5000 5000 8.2 7.3 STIM UNSTIM 4000 4000 3000 3000 2000 2000 1000 1000 0 00 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 Assessing intra-assay variability A single sample of stimulated or unstimulated human PBMC was divided into 7 tubes and assessed for expression of RPL13a mRNA in total lymphocytes (Figure 5B) or IFNγ mRNA in IFNγ + events (Figure 5A). Data shown are the average of the 7 replicates, and error bars represent standard deviation. Values shown above the bars represent the CV%. To assess the effect of target probe handling on assay variability, a sample of mouse splenocytes was divided into three samples and assayed for expression of ß-actin, RPL13a, ß2-microglobulin (B2M), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Peptidylprolyl cis-trans isomerase B (PPIB). Target probes were diluted independently for each triplicate. As shown below, the CV% were typically less than 10%, while RPL13a was slightly higher, around 12%. Intra-Experiment Variability by Probe Set 14% 14% 12% 12% CV% CV 10% 10% 8% 8% 6% 6% 4% 4% 2% 2% 0% 0% Actin Actin Actin Actin PPIB Actin Actin RPL13aRPL13aRPL13a RPL13α RPL13α RPL13α B2M B2M GAPDH GAPDH PPIB Type 11 Type Type 4 4 Type Type 66 Type Type 11 Type Type 44 Type Type 56 Type Type 44 Type Type 44 Type Type 11 Type AF647 AF488 AF647 AF647 AF488 AF488 AF488 AF647 AF647AF488AF750AF647AF488AF750AF488AF488 AF647 AF=Alexa Fluor® Figure 6: Contribution of target probe dilution to assay variability Splenocytes from C57Bl/6 mice were assessed for expression of several positive control genes. Samples were prepared in triplicate with independent dilutions of target probes made for each sample. CV% of the median fluorescence intensity for each gene are shown. 4 Operator variability To assess the contribution of technicians to assay variability, aliquots of U937 cells from the same cell culture were tested in triplicate for GAPDH, PPIB, and B2M gene expression by two different technicians. No Probe (NP) controls were used as negative controls, and samples were analyzed using the same cytometer settings. For each operator, the CV% of the mean fluorescence intensity (MFI) of triplicate samples was less than 6%, consistent with previous data for intra-assay variability. The variation between operators was approximately 5% for the MFI, and 20% for the signal-to-noise ratio, calculated as the ratio between the positive MFI and the No Probe control MFI. Figure 7B Figure 7A Operator 1 GAPDH-Type 4 Operator 2 Operator 1 GAPDH-Type 4 Operator 2 500 400 Count 350 300 MFI Count Count Count Count 450 250 200 PPIB-Type®1 Type 4 GAPDH Alexa Fluor 488 150 PPIB-Type 1 100 50 NP GAPDH NP PPIB NP B2M NP GAPDH NP PPIB NP B2M Type 4 Type 4Type 1 Type 1 Type 6 Type 6 Type 4Type 4 Type 1 T ype 1 Type 6Type 6 AF488 AF488AF647AF647 AF750AF750AF488AF488AF647AF647AF750AF750 Count Count Count Count Count 0 *AF = Alexa Fluor® Type 1 PPIB Alexa Fluor® 647 B2M-Type6 6 B2M-Type FL4 red FL4 red FL4 red Count Count Count Count Count Count FL4 red Type 6 B2M Alexa Fluor® 750 FL5red red FL5 FL5red red FL5 Contribution of operator variability U937 cells were assessed for expression of several positive control genes. Samples were prepared in triplicate by two technicians. Histogram overlays of the triplicates for No Probe and target probes are shown on the left (Figure 7A). MFI for each sample are shown on the right (Figure 7B). 5 Additional studies to assess variation between operators were run with samples containing only a subpopulation of positive cells. C57Bl/6 splenocytes were stimulated for three days with anti-CD3 and anti-CD28 antibodies, and assessed for expression of Ki-67 and Granzyme B mRNA. The samples were divided among four technicians who independently performed the assay. As shown below, the mean percentage of positive events for Ki-67 or Granzyme B were 18.6% and 30.6%, respectively (Figure 8A). The MFI of the mRNA positive events are shown in the bar graph, and in this case the CV was 12-15% (Figure 8B), consistent with the results obtained with positive control genes in U937 cells (Figure 7A & 7B). Figure 8A Count Count Contribution of operator variability in a bimodal expression model Mouse splenocytes were stimulated with Anti-Mouse CD3 (cat. no. 16-0031) and Anti-Mouse CD28 (cat. no. 16-0281) Functional Grade Purified antibodies for 3 days, with the addition of brefeldin A and monensin in the last 2 hours. The cells were stained with Anti-Mouse CD8a PE-eFluor® 610 (cat. no. 61-0081), Anti-Mouse Ki-67 eFluor® 450 (cat. no. 48-5698), and Anti-Mouse Granzyme B PE-Cyanine7 (cat. no. 25-8898), followed by hybridization of Type 4 Mouse Ki-67 Alexa Fluor ® 488 probe set (cat. no. VB4-16518) and Type 6 Mouse Granzyme B Alexa Fluor® 750 probe set (cat. no. VB6-16522). Cells in the lymphocyte gate or the mRNA+ events were used for analysis. 30.6±7.6% positive 18.6±7.1% positive Ki-67 mRNA Alexa Fluor® 488 Granzyme B mRNA Alexa Fluor® 750 Figure 8B 4500 4000 CV=15.0% Tech 1 Tech 2 MFI of mRNA+ events 3500 Tech 3 3000 Tech 4 CV=12.6% 2500 2000 1500 1000 500 0 Ki-67 Granzyme B Day-to-day variability To understand day-to-day variation, human PBMC stimulated with the Cell Stimulation Cocktail (plus protein transport inhibitor cocktail) were analyzed fresh, or cryopreserved and analyzed one week later. As shown below, the percentage of positive events is virtually unchanged between the fresh and cryopreserved samples. Day-to-day variation of PrimeFlow™ RNA Assaya IFNγ mRNAb IFNγ proteinb Experiment 1 58.0 90.3 Experiment 2 57.8 87.7 Average 57.9 89.0 Standard Deviation 0.14 1.84 CV (%) 0.24 2.07 a Normal human PBMC were stimulated and analyzed immediately (Experiment 1) or were cryopreserved and analyzed one week later (Experiment 2) for the expression of IFNγ mRNA and protein. Samples were surface stained with Anti-Human CD8a PE-eFluor® 610 (cat. no. 61-0088) and followed by hybridization of human IFNγ Alexa Fluor® 750 (cat. no. VA6-13121). b 6 Data represent the percentage of IFNγ+ events, gated on viable CD8+ cells. Sensitivity and specificity The central dogma of molecular biology states that DNA gives rise to RNA, which in turn gives rise to protein. However, studies have shown that the correlation between levels of RNA and protein products vary widely. Here we demonstrate how PrimeFlow™ RNA Assay can reveal the unique kinetics of mRNA and protein in the same cells to understand the correlation between the two over time, and in response to stimulation. Human PBMC were stimulated with the Cell Stimulation Cocktail (plus protein transport inhibitors) for as long as five hours. Using PrimeFlow RNA Assay, the cells were assessed for IFNγ or TNFα mRNA expression and protein in CD8+ or CD8- lymphocytes at hourly intervals. As shown below, both CD8+ and CD8- lymphocytes respond to stimulation, but IFNγ and TNFα mRNA and protein each exhibit unique kinetics depending on the lymphocyte subset being analyzed. Of note, while IFNγ mRNA is rapidly upregulated after one hour of stimulation, IFNγ protein is not detected until the second hour. In contrast, TNFα mRNA and protein are both upregulated within the first hour, and although CD8+ cells maintain TNFα mRNA, the CD8- lymphocytes more rapidly downregulate TNFα mRNA, and show a subsequent slow decline in TNFα protein levels. Thus, PrimeFlow RNA Assay enables the study of gene expression at the single-cell level in heterogeneous samples without the need for sorting specific subsets and has the ability to elucidate the kinetics of mRNA and protein expression. IFNγ Protein eFluor® 450 IFNγ Protein eFluor® 450 protein CD8+CD8+ protein mRNA CD8+CD8+ mRNA CD8- protein CD8- protein CD8- mRNA CD8- mRNA IFNγ Protein eFluor® 450 Figure 9B TNFα mRNA Alexa Fluor® 488 TNFα mRNA Alexa Fluor® 488 5 hr TNFα Protein PE-Cyanine7 TNFα Protein PE-Cyanine7 5 hr IFNγ Protein eFluor® 450 TNFα mRNA and protein expression in T cell subsets by FlowRNA 100 TNFα mRNA and protein expression in T cell subsets by FlowRNA 100 75 75 50 50 25 25 0 3 IFNγ Hours post-smulaon ® 0 1 IFNγ0 Protein eFluor 4502 Figure 9C 100 TNFα mRNA Alexa Fluor® 488 IFNγ Protein eFluor® 450 0 TNFα mRNA and protein expression in T cell subsets by FlowRNA TNFα mRNA Alexa Fluor® 488 4 hr IFNγ mRNA Alexa Fluor® 750 IFNγ Protein eFluor® 450 TNFα Protein PE-Cyanine7 Percent posive cells 3 hr IFNγ mRNA Alexa Fluor® 750 TNFα Protein PE-Cyanine7 TNFα Protein PE-Cyanine7 Percent posive cells TNFα mRNA Alexa Fluor® 488 TNFα mRNA Alexa Fluor® 488 TNFα Protein PE-Cyanine7 4 hr IFNγ mRNA Alexa Fluor® 750 IFNγ Protein eFluor® 450 IFNγ Protein eFluor® 450 3 hr IFNγ mRNA Alexa Fluor® 750 IFNγ Protein eFluor® 450 2 hr IFNγ mRNA Alexa Fluor® 750 IFNγ Protein eFluor® 450 TNFα Protein PE-Cyanine7 IFNγ mRNA Alexa Fluor® 750 IFNγ mRNA Alexa Fluor® 750 1 hr TNFα mRNA Alexa Fluor® 488 TNFα Protein PE-Cyanine7 TNFα Protein PE-Cyanine7 IFNγ mRNA Alexa Fluor® 750 TNFα Protein PE-Cyanine7 IFNγ mRNA Alexa Fluor® 750 IFNγ mRNA Alexa Fluor® 750 IFNγ mRNA Alexa Fluor® 750 CD8- TNFα Protein PE-Cyanine7 2 hr IFNγ mRNA Alexa Fluor® 750 TNFα mRNA Alexa Fluor® 488 TNFα Protein PE-Cyanine7 0 hr CD8+ 1 hr TNFα mRNA Alexa Fluor® 488 CD8- TNFα mRNA Alexa Fluor® 488 CD8+ TNFα mRNA Alexa Fluor® 488 0 hr TNFα mRNA Alexa Fluor® 488 Figure 9A 1 4 5 ® 450 Protein eFluor 2 3 Hours post-smulaon 4 5 IFNγ mRNA and protein expression in T cell subsets by FlowRNA 7525 500 0 1 2 3 Hours post-smulaon 4 CD8+ protein CD8+ mRNA protein CD8+CD8protein CD8- mRNA CD8+ mRNA CD8- protein CD8- mRNA 5 25 Percent posive cells 50 IFNγ mRNA and protein expression in T cell subsets by FlowRNA 75 100 Percent posive cells CD8+ mRNA CD8- protein CD8- mRNA 100 Percent posive cells CD8+ protein CD8+ mRNA CD8- protein mRNA CD8+CD8protein Percent posive cells 100 75 TNFα mRNA and protein expression in T cell subsets by FlowRNA 50 75 25 50 0 0 1 2 3 Hours post-smulaon 25 4 5 IFNγ mRNA and protein expression in T cell subsets by FlowRNA 100 0 0 0 1 2 3 Hours post-smulaon 4 5 0 1 2 3 4 5 CD8- protein CD8- mRNA Percent posive cells Percent posive cells CD8+ protein 75 Hours post-smulaon CD8+ mRNA 50 CD8- protein IFNγ mRNA and protein expression in T cell subsets by FlowRNA Correlation andCD8kinetics TNFα transcription and translation mRNAof IFNγ and 10025 Normal human PBMC were stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors) (cat. no. 00-4975) for 0-5 hrs, then subjected to PrimeFlow™ RNA Assay. Cells were intracellularly stained with Anti-Human CD8α PE-eFluor® 610 (cat. no. 61-0088), Anti-Human IFNγ eFluor® 450 (cat. no. 48-7319), and Anti-Human TNFα PE-Cyanine7 0 CD8+ protein 75 0 1 Human 2 TNFα 3 Alexa4 Fluor ®5 488 probe set (cat. no. VA4-10289) (Figure 9B) and Human IFNγ Alexa Fluor ® 750 probe set (cat. no. 25-7439), followed by hybridization with Hours post-smulaon (cat. no. VA6-13121) (Figure 9C). CD8+ or CD8- cells in the lymphocyte gate were used for analysis. CD8+ mRNA 50 25 7 Orthogonal validation PrimeFlow™ RNA Assay was assessed by examination of genes previously measured by QuantiGene® Plex Assay, a hybridization-based assay using the xMAP® Luminex® magnetic bead platform. As shown in Figure 10A, GAPDH showed the highest MFI in U937 cells, consistent with the highest relative signal captured in QuantiGene Plex Assay. HMBS had the lowest MFI, consistent with gene expression at low levels in U937 cells. Signal-to-noise ratios were calculated relative to the No Probe control sample and are shown in Figure 9B. Next, we assessed the expression of the same genes in normal human lymphocytes and monocytes (Figure 9C). Although U937 is a monocytic cell line, there were notable differences between primary monocytes and U937 cells. HMBS was completely undetectable in primary human monocytes, and instead of GAPDH, B2M was the most highly expressed gene in monocytes. Primary human lymphocytes diverged even further with very low levels of GAPDH and high levels of B2M. These data highlight the importance of understanding gene expression levels in the cells of interest, as expression can vary even in cell lines derived from the same primary cell type. Furthermore, based on QuantiGene Plex data, U937 cells are known to express 5-10 copies of HMBS mRNA, suggesting that in a fully optimized system it is possible to detect 5-10 RNA copies by PrimeFlow RNA Assay. HMBS B2M PPIB PPIB B2M PPIB GAPDHGAPDH Count Count Count Count GAPDH No Probe HMBS B2M PPIB GAPDH 1400 140.00 AlexaAlexa FluorFluor 647 647 AlexaFluor Fluor ® 647 Alexa 647 Figure 10C Lymphocytes Lymphocytes Lymphocytes Monocytes Monocytes Monocytes Monocytes 1000 100.00 800 80.00 68.9 56.1 60.00 600 400 40.00 200 20.00 1.0 3.6 0 No Probe dapB HMBS B2M PPIB GAPDH Count Count Count Count Count Count Count Count Lymphocytes 122.4 1200 120.00 Signal to Noise Ratio HMBS HMBS No probe No probe B2M No probe Figure 10B U937U937 U937 U937 Signal-to-noise ratio Figure 10A 0 NoProbe probe No HMBS HMBS B2M B2M PPIBPPIB GAPDHGAPDH Orthogonal validation of QuantiGene® Plex and PrimeFlowTM RNA Assays U937 (Figures 9A and 9B) or normal human PBMC (Figure 9C) were hybridized to a series of Type 1 Positive Control Alexa Fluor® 647 probe sets, following PrimeFlow™ RNA Assay protocol. The signal-to-noise ratio was calculated as the ratio between the MFI of the Positive Control gene and the MFI of the No Probe control. AlexaAlexa Fluor 647®647 Fluor Alexa Fluor 647 Alexa Fluor® 647 Alexa Fluor 647 Alexa Fluor 647 Alexa Fluor 647 Alexa Fluor 647 Correlation of QuantiGene® Plex and TaqMan® data To demonstrate the accuracy of QuantiGene® Plex assay, measurements of twenty transcripts from two reference RNA samples were made and compared to QuantiGene® and TaqMan® data. Reference RNA samples include human brain total RNA and universal human reference RNA as described in Canales et al. 2006. Nature Biotechnology 24(9):1115-1122. As shown below (Figure 11), excellent correlation is noted in RNA level fold changes in the reference samples, between QuantiGene® Plex and TaqMan® assays (R2 = 0.965). TaqMan® data are described in Canales, et al. 2006 publication. These studies show that TaqMan® based PCR, QuantiGene® Plex, and PrimeFlow™ RNA Assay provide consistent results. R2=0.965 Figure 11: Comparison of QuantiGene® and TaqMan® RNA level fold changes in total and reference RNA. 4 3 TaqMan® 2 1 -4 -2 -1 2 -2 -3 -4 -5 QuantiGene® Plex Assay 8 4 Technology specificity The bDNA technology achieves high target specificity with the use of oligonucleotide pairs—a design resulting in signal amplification only when two adjacent target probe oligonucleotides (left oligonucleotide and right oligonucleotide) bind to the specific target. To determine the assay specificity, probe sets for human GAPDH containing left oligonucleotides alone, or right oligonucleotides alone, were tested and compared to a complete probe set in human U937 cells. Fluorescent signal is detected only with the complete probe set, correlating to the abundant expression of GAPDH in U937 cells. In contrast, no signal is detected when either the left or right oligonucleotides are used individually, similar to the No Probe control. Additionally, a negative control probe set for DapB, a bacterial gene in Bacilus subtillis, yielded no specific signal. Figure 12 Figure 13 U937 Label Probe Label Probe Pre-amplifier Pre-Amplifier Count Count Amplifier Amplifier 800 GAPDH Left + Right GAPDH Left Oligos Only GAPDH Right Oligos Only dapB No Probe 600 400 200 RNA RNA Left Oligo Left Oligo 0 100 RightOligo Oligo Right 101 102 103 Fluorescent Signal Fluorescent signal 104 Figure 14 1200 1200 MFI MFI 1000 1000 800 800 600 600 400 400 200 200 0 0 NoProbe probe No Oligos Only Oligos Only GAPDH Left GAPDHRight Right GAPDHLeft Left GAPDH Left GAPDH GAPDH Oligos Only Oligos Only + Right Oligos Oligos Only Oligos Only +Right Oligos Dapβ DapB Demonstration of specificity of the PrimeFlow™ RNA Assay Illustration of a Probe Set design containing both left and right oligonucleotide pairs, and signal amplifications reagents, Pre-amplifier, Amplifier, and Lable Probes (Figure 12). Flow cytometry histogram data of GAPDH RNA expression in U937 cells (Figure 13). Quantitative representation of GAPDH signal with different probe sets based on Mean Fluorescent Intensity (MFI) (Figure 14). 9 Assay channel sensitivity PrimeFlow™ RNA Assay is capable of detecting three different RNA targets in a single cell through the use of three different fluorochromes. However, each fluorochrome provides different levels of sensitivity. To understand the relative sensitivity of the three RNA detection channels, we assessed the expression level of the same gene in each of the three probe set types using U937 samples or in mouse splenocytes. Human B2M probe sets in Type 1 (Alexa Fluor® 647), Type 4 (Alexa Fluor® 488), or Type 6 (Alexa Fluor® 750) were used to detect B2M RNA targets in human U937 cells (Figure 15A). The signal-to-noise ratio relative to the No Probe control was calculated and the relative sensitivity to Type 6 (Alexa Fluor 750) probe set was assessed. As shown in Figures 15A and 15B, Type 1 (Alexa Fluor 647) provides the greatest sensitivity. We observed similar sensitivity when assessing the expression of β-Actin in mouse splenocytes. For this reason, Type 1 probes for genes with low or unknown levels of expression. Type 4 (Alexa Fluor 488) and Type 6 (Alexa Fluor 750) probe sets have similar sensitivity and are recommended for genes with medium to high levels of expression. As with any multi color flow cytometry experiment, actual results depend on instrument configuration, detector sensitivity, instrument settings, and compensation, all of which require optimization to obtain the best results. Human B2M mRNA relative sensitivity (%) Figure 15A 600% 600% Assessing assay channel sensitivity U937 cells were hybridized with B2M probe sets in each probe set type to assess the different sensitivity of each channel. Values above each bar represent the signal-to-noise ratio. Total viable cells were used for analysis (Figure 15A). Mouse splenocytes were hybridized with β-Actin (ACTB) probes, in triplicate. Total viable cells were used for analysis (Figure 15B). 58.1 500% 500% 400% 400% 300% 300% 200% 200% 13.4 100% 100% 10.7 0%0% Type 1 AF647 Type 4 AF488 Type 6 AF750 AF=Alexa Fluor® Figure 15B Alexa FluorAlexa 647 Fluor 647Fluor 647 Alexa ACTB mRNA 10 Alexa FluorAlexa 647 Fluor 647Fluor 647 Alexa Count Count Count Alexa Fluor® 750 Count Count Count Count Alexa Fluor® 488 Count Count Count Count Count Alexa Fluor® 647 Alexa FluorAlexa 647 Fluor 647Fluor 647 Alexa Sample types tested PrimeFlow™ RNA Assay has been validated for use with suspension cells such as peripheral blood mononuclear cells (cryopreserved, stimulated cells and freshly isolated cells), mouse bone marrow cells, mouse tissue (fresh, stimulated and cryopreserved), cultured mammalian leukemic cell lines, and adherent cells. Cell Recommended Positive Control Gene Human lymphocytes (PBMC) – fresh and cryopreserved RPL13a, B2M Human monocytes (PBMC) – fresh and cryopreserved RPL13a, B2M Mouse splenocytes (tissue) – fresh and cryopreserved ACTB, RPL13a Mouse thymocytes (tissue) ACTB, RPL13a Mouse bone marrow ACTB Human monocytic lymphoma, U937 RPL13a, B2M Human T cell lymphoma, Jurkat RPL13a, B2M Human cervical carcinoma, HeLa* RPL13a, GAPDH Human lung carcinoma, PC9* RPL13a, GAPDH *adherent cells Adherent cell data Figure 16: Human cervical carcinoma cells, HeLa vs Human lung adenocarcinoma cells, PC9 Adherent cell lines, HeLa and PC9, were detached using either EDTA or typrsin and then subjected to PrimeFlow™ RNA Assay. HeLa and PC9 cells were hybridized with human KRT19, PPIB, and GAPDH probe sets. Helical human cervical carcinoma cells, HeLa vs human lung adenocarcinoma cells, PC9 *adherent cells 120 120.00 HeLa-Trypsin HeLa-EDTA PC9-Trypsin PC9-EDTA Signal-to-noise ratio 100 100.00 80 80.00 60 60.00 40 40.00 20 20.00 0 0.00 KRT19 KRT19 PPIB PPIB GADPH GAPDH Helical human cervical carcinoma cells, HeLa vs human lung adenocarcinoma cells, PC9 11 eBioscience Tel: +1-888-999-1371 Affymetrix, Inc. Tel: +1-888-362-2447 Tel: +1-858-642-2058 eBioscience (EU) Tel: +43 1 796 40 40 305 Affymetrix UK Ltd. Tel: +44-(0)1628-552550 Panomics Solutions Tel: +1-877-726-6642 panomics.affymetrix.com [email protected] Affymetrix Japan K.K. Tel: +81-(0)3-6430-4020 USB Products Tel: +1-800-321-9322 usb.affymetrix.com www.ebioscience.com Please visit our website for international distributor contact information. For Research Use Only. Not for use in diagnostic or therapeutic procedures. FC04271-1_PrimeFlow™ RNA Assay Technology Validation Paper 1014 ©Affymetrix, Inc. All rights reserved. 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