Simultaneous, Single‐Cell Measurement of Messenger RNA, Cell Surface Proteins, and Intracellular Proteins

Kah Teong Soh1, Joseph D. Tario1, Sean Colligan1, Orla Maguire1, Dalin Pan1, Hans Minderman1, Paul K. Wallace1

1 Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.45
DOI:  10.1002/0471142956.cy0745s75
Online Posting Date:  January, 2016
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Abstract

Nucleic acid content can be quantified by flow cytometry through the use of intercalating compounds; however, measuring the presence of specific sequences has hitherto been difficult to achieve by this methodology. The primary obstacle to detecting discrete nucleic acid sequences by flow cytometry is their low quantity and the presence of high background signals, rendering the detection of hybridized fluorescent probes challenging. Amplification of nucleic acid sequences by molecular techniques such as in situ PCR have been applied to single‐cell suspensions, but these approaches have not been easily adapted to conventional flow cytometry. An alternative strategy implements a Branched DNA technique, comprising target‐specific probes and sequentially hybridized amplification reagents, resulting in a theoretical 8,000‐ to 16,000‐fold increase in fluorescence signal amplification. The Branched DNA technique allows for the quantification of native and unmanipulated mRNA content with increased signal detection and reduced background. This procedure utilizes gentle fixation steps with low hybridization temperatures, leaving the assayed cells intact to permit their concomitant immunophenotyping. This technology has the potential to advance scientific discovery by correlating potentially small quantities of mRNA with many biological measurements at the single‐cell level. © 2016 by John Wiley & Sons, Inc.

Keywords: flow cytometry; Branched DNA; mRNA sequence; target probe; in situ hybridization; leukocytes; transcription factors

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of mRNA Transcripts in PBMCs Using the Branched DNA Assay
  • Support Protocol 1: Temperature Validation for the Hybridization Oven to Achieve and Maintain 40° ± 1°C
  • Alternate Protocol 1: Simultaneous Measurement of mRNA Transcripts in, and Cell Surface Proteins on PBMCs Using the Branched DNA Assay
  • Support Protocol 2: Effect of the Branched DNA Technique on the Measurement of Different CD Antigen Clones and Fluorochromes
  • Support Protocol 3: Kinetics of CD8 mRNA in, and CD8 Protein Expression on PBMCs after α‐CD3 and α‐CD28 Activation
  • Alternate Protocol 2: Simultaneous Measurement of mRNA Transcripts and Intracellular Proteins in PBMCs Using the Branched DNA Assay
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of mRNA Transcripts in PBMCs Using the Branched DNA Assay

  Materials
  • Specimen: fresh EDTA‐ or sodium heparin–anticoagulated human blood, bone marrow, or leukoreduction filter retentate specimens may be used in this protocol; samples should be stored at room temperature <48 hr (see Tario et al., , for a discussion on the preparation of PBMCs from leukoreduction filter retentates)
  • Hanks’ Balanced Salt Solution (HBSS; Corning, cat. no. 21‐023‐CV; store at 2° to 8°C)
  • PrimeFlow RNA kit (Affymetrix, cat. no. 88‐18009‐210) containing the following:
    • 1.5‐ml Branched DNA Microcentrifuge Labeling Tubes (microcentrifuge tubes)
    • Branched DNA Fixation Buffer 1A (2×; store at 2° to 8°C)
    • Branched DNA Fixation Buffer 1B (2×; store at 2° to 8°C)
    • Branched DNA Permeabilization Buffer (10×; store at 2° to 8°C)
    • RNase Inhibitor (1000×; store at –20°C)
    • Branched DNA Fixation Buffer 2 (8×; store at 2° to 8°C)
    • Branched DNA Wash Buffer (1×; store at 2° to 8°C)
    • Positive Control Target Probe Sets (20×; one for each mRNA detection channel; store at –20°C)
    • Branched DNA Target Probe Diluent (store at 2° to 8°C)
    • Branched DNA PreAmp Mix (store at 2° to 8°C)
    • Branched DNA Amp Mix (store at 2° to 8°C)
    • Branched DNA Label Probe Diluent (store at 2° to 8°C)
    • Branched DNA Label Probes (100×; store at –20°C)
    • Branched DNA Storage Buffer (store at 2° to 8°C)
  • DNase‐, RNase‐, and protease‐free H 2O (RNase‐free H 2O; molecular biology grade; Corning, cat. no. 46‐000‐CV)
  • Target Probe(s) specific to mRNA sequence(s) of interest: in this example, CD8 mRNA Alexa Fluor 647 probes (Type 1, Affymetrix, cat. no. VA1‐16494) and RPL13A Alexa Fluor 750 (Type 6, Affymetrix, cat. no. VA6‐13186) are employed; custom target probe sets for specific mRNA target sequences of interest can be obtained from the manufacturer (tech@ebioscience.com); store at –20°C
  • Flow cytometry staining (FCM) buffer (see recipe)
  • 15‐ and/or 50‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Centrifuge
  • Vacuum aspiration device
  • Hybridization oven or incubator capable of achieving and maintaining a temperature of 40° ± 1°C (Affymetrix, cat. no. QS0704; also see protocol 2)
  • Digital NIST‐traceable thermometer capable of accurately measuring temperature at 40°C (Affymetrix, cat. no. QV0523; also see protocol 2)
  • 12 × 75–mm polystyrene tubes (BD Falcon, cat. no. 352052)
  • Aluminum heat block that is milled to closely fit 1.5‐ml microcentrifuge tubes (VWR, cat. no. 13259‐002)
  • Flow cytometer equipped with excitation sources and detector configurations for measuring fluorescence generated from Alexa Fluor 488, Alexa Fluor 647, and/or Alexa Fluor 750
  • Flow cytometry data analysis software package (e.g., WinList, Verity Software House; FlowJo, FlowJo LLC; FACSDiVa, BD Biosciences; or Kaluza, Beckman Coulter)
  • Additional reagents and equipment for PBMC preparation via density gradient centrifugation (Boyum, ; Castle, ; Fuss et al., ) and cell counting ( appendix 3A; Phelan and Lawler, )

Support Protocol 1: Temperature Validation for the Hybridization Oven to Achieve and Maintain 40° ± 1°C

  Additional Materials
  • Human IgG Fc Block Solution, 12 mg/ml (Sigma‐Aldrich, cat. no. I2511) diluted in RPMI 1640 (Corning, cat. no. 10‐040‐CV)
  • Pre‐titrated, fluorescently‐labeled mAbs with specificity to targets of interest—in this example:
    • CD8 BV421 (Biolegend, cat. no. 301035, Clone RPA‐T8)
    • CD14 BV510 (Biolegend, cat. no. 301841, Clone M5E2)
    • CD56 PECy7 (BD Bioscience, cat. no. 335809, Clone NCAM16.2)

Alternate Protocol 1: Simultaneous Measurement of mRNA Transcripts in, and Cell Surface Proteins on PBMCs Using the Branched DNA Assay

  Additional Materials (also see protocol 1Basic Protocol and protocol 3)
  • Refer to Table 7.45.3 for a comprehensive list of fluorochrome‐conjugated mAbs that were tested
Table 7.5.3   Additional Materials (also see protocol 1Basic Protocol and protocol 3)Fluorochrome‐Conjugated mAbs Used in the Study aExperimental Layout for the Simultaneous Measurement of Cell Surface Proteins, Intracellular Proteins, and CD8 mRNA in PBMCs Using the Branched DNA Assay

Antibody specificity Format Catalog number Clone Vendor
CD3 PE 347347 SK7 BD Biosciences
CD3 V450 652356 SK7 BD Biosciences
CD3 BV510 344827 SK7 BioLegend
CD3 FITC 349201 SK7 BD Biosciences
CD3 PE 347347 SK7 BD Biosciences
CD3 PerCPCy5.5 340948 SK7 BD Biosciences
CD3 PECy7 341101 SK7 BD Biosciences
CD3 APC 340661 SK7 BD Biosciences
CD3 APCH7 641406 SK7 BD Biosciences
CD4 PE 340670 SK3 BD Biosciences
CD4 V450 651850 SK3 BD Biosciences
CD4 BV510 562970 SK3 BD Biosciences
CD4 FITC 340133 SK3 BD Biosciences
CD4 PE 340670 SK3 BD Biosciences
CD4 PerCPCy5.5 341653 SK3 BD Biosciences
CD4 PECy7 348799 SK3 BD Biosciences
CD4 APC 340672 SK3 BD Biosciences
CD4 APCH7 641407 SK3 BD Biosciences
CD8 PE MHCD0804‐4 3B5 Thermo Fisher Scientific
CD10 PE IM1915U ALB1 Beckman Coulter
CD13 PE 340686 L138 BD Biosciences
CD14 PE 340683 MφP9 BD Biosciences
CD16 PE MHCD1604‐4 3G8 Thermo Fisher Scientific
CD20 PE 346595 L27 BD Biosciences
CD25 PE 341010 2A3 BD Biosciences
CD33 PE IM1179U D3HL60.251 Beckman Coulter
CD45RO PE IM1307 UCHL 1 Beckman Coulter
CD56 PE 6603067 NKH‐1 Beckman Coulter
CD56 PE 12‐0567‐41 CMSSB eBioscience
CD56 PECy7 25‐0567‐41 CMSSB eBioscience
CD56 PECy7 335809 NCAM16.2 BD Biosciences
CD56 FITC 340410 NCAM16.2 BD Biosciences
CD64 PE CD64‐322P 32.2 Trillium Diagnostic
CD69 PE 341652 L78 BD Biosciences
405‐nm laser 488‐nm laser 640‐nm laser
Tube BV421 BV510 PE PE‐eFluor610 PECy7 AX647 a AX750 b
number Tube description V450/50 V525/50 B575/26 B610/20 B780/60 R670/14 R780/60
1 Unlabeled c d
2 Single color controls BV421 CD8 (p) e  —
3 BV510 CD14 (p)
4 PE CD8 (p)
5 PE‐eFluor610 EOMES (p)
6 PECy7 CD56 (p)
7 AX647 RPL13A (m) f
8 AX750 RPL13A (m)
9 Fluorescence minus one controls No BV421 CD14 (p) T‐bet (p) EOMES (p) CD56 (p) CD8 (m) RPL13A (m)
10 No BV510 CD8 (p) T‐bet (p) EOMES (p) CD56 (p) CD8 (m) RPL13A (m)
11 No PE CD8 (p) CD14 (p) EOMES (p) CD56 (p) CD8 (m) RPL13A (m)
12 No PE‐eFluor610 CD8 (p) CD14 (p) T‐bet (p) CD56 (p) CD8 (m) RPL13A (m)
13 No PECy7 CD8 (p) CD14 (p) T‐bet (p) EOMES (p) CD8 (m) RPL13A (m)
14 No AX647 CD8 (p) CD14 (p) T‐bet (p) EOMES (p) CD56 (p) RPL13A (m)
15 No AX750 CD8 (p) CD14 (p) T‐bet (p) EOMES (p) CD56 (p) CD8 (m)
16 Experimental tube CD8 (p) CD14 (p) T‐bet (p) EOMES (p) CD56 (p) CD8 (m) RPL13A (m)

 aAPC, allophycocyanin; APCH7, allophycocyanin Hilite 7; BV510, Brilliant Violet 510; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PECy7, phycoerythrin cyanine 7; PerCPCy5.5, peridinin chlorophyll cyanine 5.5; V450, V450.
Table 7.5.4   Additional Materials (also see protocol 1Basic Protocol and protocol 3)Fluorochrome‐Conjugated mAbs Used in the Study aExperimental Layout for the Simultaneous Measurement of Cell Surface Proteins, Intracellular Proteins, and CD8 mRNA in PBMCs Using the Branched DNA Assay

Antibody specificity Format Catalog number Clone Vendor
CD3 PE 347347 SK7 BD Biosciences
CD3 V450 652356 SK7 BD Biosciences
CD3 BV510 344827 SK7 BioLegend
CD3 FITC 349201 SK7 BD Biosciences
CD3 PE 347347 SK7 BD Biosciences
CD3 PerCPCy5.5 340948 SK7 BD Biosciences
CD3 PECy7 341101 SK7 BD Biosciences
CD3 APC 340661 SK7 BD Biosciences
CD3 APCH7 641406 SK7 BD Biosciences
CD4 PE 340670 SK3 BD Biosciences
CD4 V450 651850 SK3 BD Biosciences
CD4 BV510 562970 SK3 BD Biosciences
CD4 FITC 340133 SK3 BD Biosciences
CD4 PE 340670 SK3 BD Biosciences
CD4 PerCPCy5.5 341653 SK3 BD Biosciences
CD4 PECy7 348799 SK3 BD Biosciences
CD4 APC 340672 SK3 BD Biosciences
CD4 APCH7 641407 SK3 BD Biosciences
CD8 PE MHCD0804‐4 3B5 Thermo Fisher Scientific
CD10 PE IM1915U ALB1 Beckman Coulter
CD13 PE 340686 L138 BD Biosciences
CD14 PE 340683 MφP9 BD Biosciences
CD16 PE MHCD1604‐4 3G8 Thermo Fisher Scientific
CD20 PE 346595 L27 BD Biosciences
CD25 PE 341010 2A3 BD Biosciences
CD33 PE IM1179U D3HL60.251 Beckman Coulter
CD45RO PE IM1307 UCHL 1 Beckman Coulter
CD56 PE 6603067 NKH‐1 Beckman Coulter
CD56 PE 12‐0567‐41 CMSSB eBioscience
CD56 PECy7 25‐0567‐41 CMSSB eBioscience
CD56 PECy7 335809 NCAM16.2 BD Biosciences
CD56 FITC 340410 NCAM16.2 BD Biosciences
CD64 PE CD64‐322P 32.2 Trillium Diagnostic
CD69 PE 341652 L78 BD Biosciences
405‐nm laser 488‐nm laser 640‐nm laser
Tube BV421 BV510 PE PE‐eFluor610 PECy7 AX647 a AX750 b
number Tube description V450/50 V525/50 B575/26 B610/20 B780/60 R670/14 R780/60
1 Unlabeled c d
2 Single color controls BV421 CD8 (p) e  —
3 BV510 CD14 (p)
4 PE CD8 (p)
5 PE‐eFluor610 EOMES (p)
6 PECy7 CD56 (p)
7 AX647 RPL13A (m) f
8 AX750 RPL13A (m)
9 Fluorescence minus one controls No BV421 CD14 (p) T‐bet (p) EOMES (p) CD56 (p) CD8 (m) RPL13A (m)
10 No BV510 CD8 (p) T‐bet (p) EOMES (p) CD56 (p) CD8 (m) RPL13A (m)
11 No PE CD8 (p) CD14 (p) EOMES (p) CD56 (p) CD8 (m) RPL13A (m)
12 No PE‐eFluor610 CD8 (p) CD14 (p) T‐bet (p) CD56 (p) CD8 (m) RPL13A (m)
13 No PECy7 CD8 (p) CD14 (p) T‐bet (p) EOMES (p) CD8 (m) RPL13A (m)
14 No AX647 CD8 (p) CD14 (p) T‐bet (p) EOMES (p) CD56 (p) RPL13A (m)
15 No AX750 CD8 (p) CD14 (p) T‐bet (p) EOMES (p) CD56 (p) CD8 (m)
16 Experimental tube CD8 (p) CD14 (p) T‐bet (p) EOMES (p) CD56 (p) CD8 (m) RPL13A (m)

 aAX647: Alexa Fluor 647.
 bAX750: Alexa Fluor 750.
 cSamples are exposed to all Branched DNA reagents, except for Target Probes.
 d() Denotes no Target Probes are added.
 eDenotes the measurement of protein expression.
 fDenotes the measurement of mRNA for the associated target sequence.

Support Protocol 2: Effect of the Branched DNA Technique on the Measurement of Different CD Antigen Clones and Fluorochromes

  Additional Materials (also see protocol 1Basic Protocol and protocol 3)
  • Unconjugated azide‐free anti‐CD3 (eBioscience, cat. no. 16‐0037, Clone OKT3)
  • Unconjugated azide‐free anti‐CD28 (eBioscience, cat. no. 16‐0289, Clone CD28.2)
  • Complete medium (see recipe)
  • 6 Peak Ultra Rainbow Fluorescent Calibration Beads, (Spherotech Inc., cat. no. URCP‐50‐2 K)
  • Zombie UV Fixable Viability Dye (BioLegend, cat. no. 423107; optional)
  • 96‐well, round‐bottom polypropylene plates (Corning, cat. no. 3365)
  • ImageStreamX Mark II imaging cytometer (EMD Millipore)

Support Protocol 3: Kinetics of CD8 mRNA in, and CD8 Protein Expression on PBMCs after α‐CD3 and α‐CD28 Activation

  Additional Materials (also see protocol 1Basic Protocol and protocol 3)
  • Pre‐titrated, fluorescently‐labeled mAbs with specificity to targets of interest—in this example: anti‐human T‐bet PE (eBioscience, cat. no. 12‐5825, Clone eBio4B10) and anti‐human EOMES PE‐eFluor610 (eBioscience, cat. no. 61‐4877, Clone WD1928)
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Literature Cited

Literature Cited
  Arroz, M., Came, N., Lin, P., Chen, W., Yuan, C., Lagoo, A., Monreal, M., de Tute, R., Vergilio, J.A., Rawstron, A.C., and Paiva, B. 2015. Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting. Cytometry B Clin. Cytom. doi: 10.1002/cyto.b.21228. [Epub ahead of print].
  Bauman, J.G. and Bentvelzen, P. 1988. Flow cytometric detection of ribosomal RNA in suspended cells by fluorescent in situ hybridization. Cytometry 9:517‐524. doi: 10.1002/cyto.990090602.
  Bayer, J.A. and Bauman, J.G. 1990. Flow cytometric detection of beta‐globin mRNA in murine haemopoietic tissues using fluorescent in situ hybridization. Cytometry 11:132‐143. doi: 10.1002/cyto.990110116.
  Bishop, J.A., Ma, X.J., Wang, H., Luo, Y., Illei, P.B., Begum, S., Taube, J.M., Koch, W.M., and Westra, W.H. 2012. Detection of transcriptionally active high‐risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method. Am. J. Surg. Pathol. 36:1874‐1882. doi: 10.1097/PAS.0b013e318265fb2b.
  Borzi, R.M., Piacentini, A., Monaco, M.C., Lisignoli, G., Degrassi, A., Cattini, L., Santi, S., and Facchini, A. 1996. A fluorescent in situ hybridization method in flow cytometry to detect HIV‐1 specific RNA. J. Immunol. Methods 193:167‐176. doi: 10.1016/0022-1759(96)00070-1.
  Boyum, A. 1968. Isolation of leucocytes from human blood. Further observations. Methylcellulose, dextran, and ficoll as erythrocyte aggregating agents. Scand J. Clin. Lab. Invest. Suppl. 97:31‐50.
  Buckingham, L. and Flaws, M.L. 2007. Molecular Diagnostics: Fundamentals, Methods, and Clinical Applications. F.A. Davis, Philadelphia, Pa.
  Buggert, M., Tauriainen, J., Yamamoto, T., Frederiksen, J., Ivarsson, M.A., Michaelsson, J., Lund, O., Hejdeman, B., Jansson, M., Sonnerborg, A., Koup, R.A., Betts, M.R., and Karlsson, A.C. 2014. T‐bet and Eomes are differentially linked to the exhausted phenotype of CD8+ T cells in HIV infection. PLoS Pathog. 10:e1004251. doi: 10.1371/journal.ppat.1004251.
  Castle, D.C. 1998. Overview of cell fractionation. Curr. Protoc. Cell Biol. 69:3.1.1‐3.1.9.
  Chen, R.H. and Fuggle, S.V. 1993. In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression. Am. J. Pathol. 143:1527‐1534.
  Collins, M.L., Irvine, B., Tyner, D., Fine, E., Zayati, C., Chang, C., Horn, T., Ahle, D., Detmer, J., Shen, L.P., Kolberg, J., Bushnell, S., Urdea, M.S., and Ho, D.D. 1997. A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. Nucleic Acids Res. 25:2979‐2984. doi: 10.1093/nar/25.15.2979.
  Cox, J. and Mann, M. 2007. Is proteomics the new genomics? Cell 130:395‐398. doi: 10.1016/j.cell.2007.07.032.
  Fuss, I.J., Kanof, M.E., Smith, P.D., and Zola, H. 2009. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr. Protoc. Immunol. 85:7.1.1‐7.1.8.
  Gall, J.G. and Pardue, M.L. 1969. Formation and detection of RNA‐DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. U.S.A. 63:378‐383. doi: 10.1073/pnas.63.2.378.
  Garcia‐Morales, R., Carreno, M., Mathew, J., Zucker, K., Cirocco, R., Ciancio, G., Burke, G., Roth, D., Temple, D., Rosen, A., Fuller, L., Esquenazi, V., Karatzas, T., Ricordi, C., Tzakis, A., and Miller, J. 1997. The effects of chimeric cells following donor bone marrow infusions as detected by PCR‐flow assays in kidney transplant recipients. J. Clin. Invest. 99:1118‐1129. doi: 10.1172/JCI119240.
  Gibbings, D.J., Marcet‐Palacios, M., Sekar, Y., Ng, M.C., and Befus, A.D. 2007. CD8 alpha is expressed by human monocytes and enhances Fc gamma R‐dependent responses. BMC immunol. 8:12. doi: 10.1186/1471-2172-8-12.
  Gibellini, D.E., Re, M.C., Furlini, G., and La Placa, M. 1997. Flow cytometry analysis of an in situ PCR for the detection of human immunodeficiency virus type‐1 (HIV‐1) proviral DNA. Methods Mol. Biol. 71:113‐122.
  Goolsby, C.L., Thompson, E., and Mosiman, V. 2000. Combined immunophenotyping and molecular immunophenotyping. In Immunophenotyping (C.C. Stewart and J.K.A. Nicholson, eds.) pp. 407‐427. Wiley‐Liss, New York.
  Hanley, M.B., Lomas, W., Mittar, D., Maino, V., and Park, E. 2013. Detection of low abundance RNA molecules in individual cells by flow cytometry. PLoS One 8:e57002. doi: 10.1371/journal.pone.0057002.
  Harper, M.E., Marselle, L.M., Gallo, R.C., and Wong‐Staal, F. 1986. Detection of lymphocytes expressing human T‐lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc. Natl. Acad. Sci. U.S.A. 83:772‐776. doi: 10.1073/pnas.83.3.772.
  Holtke, H.J. and Kessler, C. 1990. Non‐radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA‐based detection. Nucleic Acids Res. 18:5843‐5851. doi: 10.1093/nar/18.19.5843.
  Intlekofer, A.M., Banerjee, A., Takemoto, N., Gordon, S.M., Dejong, C.S., Shin, H., Hunter, C.A., Wherry, E.J., Lindsten, T., and Reiner, S.L. 2008. Anomalous type 17 response to viral infection by CD8+ T cells lacking T‐bet and eomesodermin. Science 321:408‐411. doi: 10.1126/science.1159806.
  Kapke, G.E., Watson, G., Sheffler, S., Hunt, D., and Frederick, C. 1997. Comparison of the chiron quantiplex branched DNA (bDNA) assay and the Abbott Genostics solution hybridization assay for quantification of hepatitis B viral DNA. J. Viral. Hepat. 4:67‐75. doi: 10.1046/j.1365-2893.1997.00127.x.
  Klemm, S., Semrau, S., Wiebrands, K., Mooijman, D., Faddah, D.A., Jaenisch, R., and van Oudenaarden, A. 2014. Transcriptional profiling of cells sorted by RNA abundance. Nat. Methods 11:549‐551. doi: 10.1038/nmeth.2910.
  Knox, J.J., Cosma, G.L., Betts, M.R., and McLane, L.M. 2014. Characterization of T‐bet and eomes in peripheral human immune cells. Front Immunol. 5:217. doi: 10.3389/fimmu.2014.00217.
  Koopman, P. 2001. In situ hybridization to mRNA: From black art to guiding light. Int. J. Dev. Biol. 45:619‐622.
  Levsky, J.M. and Singer, R.H. 2003. Fluorescence in situ hybridization: Past, present and future. J Cell Sci 116:2833‐2838. doi: 10.1242/jcs.00633.
  Li, B.D., Timm, E.A., Jr., Riedy, M.C., Harlow, S.P., and Stewart, C.C. 1994. Molecular phenotyping by flow cytometry. Methods Cell Biol. 42(Pt B):95‐130. doi: 10.1016/S0091-679X(08)61070-5.
  Liang, Y.J., Lao, X.M., Liang, L.Z., and Liao, G.Q. 2015. Genome‐wide analysis of cancer cell‐derived Foxp3 target genes in human tongue squamous cell carcinoma cells. Int. J. Oncol. 46:1935‐1943.
  McLane, L.M., Banerjee, P.P., Cosma, G.L., Makedonas, G., Wherry, E.J., Orange, J.S., and Betts, M.R. 2013. Differential localization of T‐bet and Eomes in CD8 T cell memory populations. J. Immunol. 190:3207‐3215. doi: 10.4049/jimmunol.1201556.
  Minderman, H., Humphrey, K., Arcadi, J.K., Wierzbicki, A., Maguire, O., Wang, E.S., Block, A.W., Sait, S.N., George, T.C., and Wallace, P.K. 2012. Image cytometry‐based detection of aneuploidy by fluorescence in situ hybridization in suspension. Cytometry A 81:776‐784. doi: 10.1002/cyto.a.22101. Epub 2012 Jul 26.
  Mutty, C.E., Timm, E.A., Jr., and Stewart, C.C. 1999. Effects of thermal exposure on immunophenotyping combined with in situ PCR, measured by flow cytometry. Cytometry 36:303‐311. doi: 10.1002/(SICI)1097-0320(19990801)36:4%3c303::AID-CYTO4%3e3.0.CO;2-.
  Neale, G.A., Coustan‐Smith, E., Stow, P., Pan, Q., Chen, X., Pui, C.H., and Campana, D. 2004. Comparative analysis of flow cytometry and polymerase chain reaction for the detection of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia 18:934‐938. doi: 10.1038/sj.leu.2403348.
  Patterson, B.K., Goolsby, C., Hodara, V., Lohman, K.L., and Wolinsky, S.M. 1995. Detection of CD4+ T cells harboring human immunodeficiency virus type 1 DNA by flow cytometry using simultaneous immunophenotyping and PCR‐driven in situ hybridization: Evidence of epitope masking of the CD4 cell surface molecule in vivo. J. Virol. 69:4316‐4322.
  Patterson, B.K., Mosiman, V.L., Cantarero, L., Furtado, M., Bhattacharya, M., and Goolsby, C. 1998. Detection of HIV‐RNA‐positive monocytes in peripheral blood of HIV‐positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype. Cytometry 31:265‐274. doi: 10.1002/(SICI)1097-0320(19980401)31:4%3c265::AID-CYTO6%3e3.0.CO;2-I.
  Patterson, B.K., Till, M., Otto, P., Goolsby, C., Furtado, M.R., McBride, L.J., and Wolinsky, S.M. 1993. Detection of HIV‐1 DNA and messenger RNA in individual cells by PCR‐driven in situ hybridization and flow cytometry. Science 260:976‐979. doi: 10.1126/science.8493534.
  Phelan, M.C. and Lawler, G. 1997. Cell counting. Curr. Protoc. Cytom. 00:A.3A.1‐A.3A.4.
  Player, A.N., Shen, L.P., Kenny, D., Antao, V.P., and Kolberg, J.A. 2001. Single‐copy gene detection using branched DNA (bDNA) in situ hybridization. J. Histochem. Cytochem. 49:603‐612. doi: 10.1177/002215540104900507.
  Porichis, F., Hart, M.G., Griesbeck, M., Everett, H.L., Hassan, M., Baxter, A.E., Lindqvist, M., Miller, S.M., Soghoian, D.Z., Kavanagh, D.G., Reynolds, S., Norris, B., Mordecai, S.K., Nguyen, Q., Lai, C., and Kaufmann, D.E. 2014. High‐throughput detection of miRNAs and gene‐specific mRNA at the single‐cell level by flow cytometry. Nat. Commun. 5:5641. doi: 10.1038/ncomms6641.
  Roederer, M. 2001. Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats. Cytometry 45:194‐205. doi: 10.1002/1097-0320(20011101)45:3%3c194::AID-CYTO1163%3e3.0.CO;2-C.
  Rondeau, S., Vacher, S., De Koning, L., Briaux, A., Schnitzler, A., Chemlali, W., Callens, C., Lidereau, R., and Bieche, I. 2015. ATM has a major role in the double‐strand break repair pathway dysregulation in sporadic breast carcinomas and is an independent prognostic marker at both mRNA and protein levels. Br. J. Cancer 112:1059‐1066. doi: 10.1038/bjc.2015.60.
  Tario, J.D., Jr., Muirhead, K.A., Pan, D., Munson, M.E., and Wallace, P.K. 2011. Tracking immune cell proliferation and cytotoxic potential using flow cytometry. Methods Mol. Biol. 699:119‐164. doi: 10.1007/978-1-61737-950-5_7.
  Trask, B., van den Engh, G., Landegent, J., in de Wal, N.J., and van der Ploeg, M. 1985. Detection of DNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry. Science 230:1401‐1403. doi: 10.1126/science.2416058.
  Tsukamoto, T., Kusakabe, M., and Saga, Y. 1991. In situ hybridization with non‐radioactive digoxigenin‐11‐UTP‐labeled cRNA probes: Localization of developmentally regulated mouse tenascin mRNAs. Int. J. Dev. Biol. 35:25‐32.
  Tyakht, A.V., Ilina, E.N., Alexeev, D.G., Ischenko, D.S., Gorbachev, A.Y., Semashko, T.A., Larin, A.K., Selezneva, O.V., Kostryukova, E.S., Karalkin, P.A., Vakhrushev, I.V., Kurbatov, L.K., Archakov, A.I., and Govorun, V.M. 2014. RNA‐Seq gene expression profiling of HepG2 cells: The influence of experimental factors and comparison with liver tissue. BMC Genomics 15:1108. doi: 10.1186/1471-2164-15-1108.
  Ukpo, O.C., Flanagan, J.J., Ma, X.J., Luo, Y., Thorstad, W.L., and Lewis, J.S., Jr. 2011. High‐risk human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma. Am. J. Surg. Pathol. 35:1343‐1350. doi: 10.1097/PAS.0b013e318220e59d.
  Unwin, R.D. and Whetton, A.D. 2006. Systematic proteome and transcriptome analysis of stem cell populations. Cell Cycle 5:1587‐1591. doi: 10.4161/cc.5.15.3101.
  van Dekken, H., Arkesteijn, G.J., Visser, J.W., and Bauman, J.G. 1990. Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei. Cytometry 11:153‐164. doi: 10.1002/cyto.990110118.
  Van Hoof, D., Lomas, W., Hanley, M.B., and Park, E. 2014. Simultaneous flow cytometric analysis of IFN‐gamma and CD4 mRNA and protein expression kinetics in human peripheral blood mononuclear cells during activation. Cytometry A 85:894‐900. doi: 10.1002/cyto.a.22521.
  Walter, R.F., Mairinger, F.D., Ting, S., Vollbrecht, C., Mairinger, T., Theegarten, D., Christoph, D.C., Schmid, K.W., and Wohlschlaeger, J. 2015. MDM2 is an important prognostic and predictive factor for platin‐pemetrexed therapy in malignant pleural mesotheliomas and deregulation of P14/ARF (encoded by CDKN2A) seems to contribute to an MDM2‐driven inactivation of P53. Br. J. Cancer 112:883‐890. doi: 10.1038/bjc.2015.27.
  Wang, F., Flanagan, J., Su, N., Wang, L.C., Bui, S., Nielson, A., Wu, X., Vo, H.T., Ma, X.J., and Luo, Y. 2012. RNAscope: A novel in situ RNA analysis platform for formalin‐fixed, paraffin‐embedded tissues. J. Mol. Diagn. 14:22‐29. doi: 10.1016/j.jmoldx.2011.08.002.
  Weng, X.Q., Shen, Y., Sheng, Y., Chen, B., Wang, J.H., Li, J.M., Mi, J.Q., Chen, Q.S., Zhu, Y.M., Jiang, C.L., Yan, H., Zhang, X.X., Huang, T., Zhu, Z., Chen, Z., and Chen, S.J. 2013. Prognostic significance of monitoring leukemia‐associated immunophenotypes by eight‐color flow cytometry in adult B‐acute lymphoblastic leukemia. Blood Cancer J. 3:e133. doi: 10.1038/bcj.2013.31.
  Wiegant, J., Ried, T., Nederlof, P.M., van der Ploeg, M., Tanke, H.J., and Raap, A.K. 1991. In situ hybridization with fluoresceinated DNA. Nucleic Acids Res. 19:3237‐3241. doi: 10.1093/nar/19.12.3237.
  Yang, G., Garhwal, S., Olson, J.C., and Vyas, G.N. 1994. Flow cytometric immunodetection of human immunodeficiency virus type 1 proviral DNA by heminested PCR and digoxigenin‐labeled probes. Clin. Diagn. Lab. Immunol. 1:26‐31.
  Yang, G., Olson, J.C., Pu, R., and Vyas, G.N. 1995. Flow cytometric detection of human immunodeficiency virus type 1 proviral DNA by the polymerase chain reaction incorporating digoxigenin‐ or fluorescein‐labeled dUTP. Cytometry 21:197‐202. doi: 10.1002/cyto.990210212.
  Yu, H., Ernst, L., Wagner, M., and Waggoner, A. 1992. Sensitive detection of RNAs in single cells by flow cytometry. Nucleic Acids Res. 20:83‐88. doi: 10.1093/nar/20.1.83.
  Zhang, L., Zhou, W., Velculescu, V.E., Kern, S.E., Hruban, R.H., Hamilton, S.R., Vogelstein, B., and Kinzler, K.W. 1997. Gene expression profiles in normal and cancer cells. Science 276:1268‐1272. doi: 10.1126/science.276.5316.1268.
Key References
  Hanley et al., 2013. See above.
  This paper demonstrated for the first time the simultaneous analysis of multiple mRNAs at the sensitivity detection limit for a single mRNA transcript in individual cells. This was achieved by adapting a novel signal amplification technology (also known as Branched DNA) which suppressed background while amplifying specific target signals.
  Van Hoof et al., 2014. See above.
  This is the first study that incorporated the simultaneous and correlated measurement of mRNA and protein species by multiparamteric flow cytometry using the Branched DNA methodology.
  Porichis et al., 2014. See above.
  This paper demonstrated the potential of the Branched DNA methodology to detect mRNAs of interest, when the availability of mAb is limited. Simultaneous protein and mRNA detection was accomplished using ImageStream technology.
  Wang et al., 2012. See above.
  This article provided the technical background associated with the Branched DNA assay.
Internet Resources
  http://www.acdbio.com/products/rnascope‐assays
  Advanced Cell Diagnostics (ACD) originated RNAScope technology, which represents the foundation of the Branched DNA methodology. This signal‐amplification platform was developed for immunohistochemistry, but has been adapted by other companies for the detection of mRNA by flow cytometry.
  http://www.ebioscience.com/knowledge‐center/application/flowrna/technology.htm
  This Web site provides technical background and flow cytometric applications of the Branched DNA technology, in association with the PrimeFlow RNA Kit.
  http://www.ebioscience.com/media/newpdf/PrimeFlowRNAAssay_UM010915.pdf
  This Web site provides a PDF version of the Prime FlowRNA assay protocol, which was written by Affymetrix, Inc.
  http://www.ebioscience.com/resources/faq/flowrna‐faq.htm
  This Web site provides technical support for the PrimeFlow RNA Kit.
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