Detection of Gene Fusions in Acute Leukemia Using Bead Microarrays

German Pihan1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.7
DOI:  10.1002/0471142956.cy1307s35
Online Posting Date:  February, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes a method for the rapid and simultaneous detection of hybrid mRNAs (hRNA; also known as gene fusions, RNA fusions, or chimeric RNA), resulting from recurrent chromosome translocations or inversions in acute leukemia. The principal components of the assay are a multiplex RT‐PCR, which simultaneously amplifies any of a number of possible hRNA targets, and a liquid bead microarray (LBMA), which is capable of unambiguously identifying the amplified hRNA. The entire procedure from RNA extraction to display of results can be broken into four steps: (1) conjugated‐microsphere and LBMA manufacture and quality control, (2) cDNA synthesis, (3) multiplex PCR, and (4) hybridization of multiplex PCR products to the hRNA‐detecting LBMA and assay read‐out on a flow cytometer. The assay is designed to detect the most common risk‐stratifying translocation in pediatric acute lymphoblastic leukemia, satisfying the demand for inclusion of genetic data in the diagnosis of acute leukemia as predicated by the current WHO classification of hematopoietic and lymphoid neoplasms.

Keywords: hybrid mRNA; gene fusions; bead microarray; leukemia

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: hRNA‐LBMA Manufacture and Quality Control
  • Support Protocol 1: wLBMA Preparation and Quality Control
  • Basic Protocol 2: PCR Synthesis of cDNA Using Random Hexamer Primers
  • Alternate Protocol 1: cDNA Synthesis Using Gene‐Specific Primers or Single‐Tube RT‐PCR
  • Basic Protocol 3: Amplification of Target cDNA by Multiplex PCR
  • Alternate Protocol 2: Exonuclease I‐Enabled ssPCR of Target cDNA
  • Basic Protocol 4: Hybridization of Multiplex PCR Products to the hRNA‐Detecting LBMA and Sample Read‐Out
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: hRNA‐LBMA Manufacture and Quality Control

  Materials
  • Carboxylated microspheres (e.g., xMAP beads, Luminex)
  • 0.1M 2‐[N‐morpholino]ethanesulfonic acid buffer (MES; Pierce), pH 4.5: prepare with sterile water, adjust pH with NaOH, and store up to 1 month at room temperature
  • 1 mM amino‐modified capture‐probe oligonucleotides (CPOs; see Table 13.7.1): prepare with sterile water and store indefinitely at −20°C
  • 10 mg/ml 1‐ethyl‐3‐[3‐dimethylaminopropyl]carbodiimide hydrochloride (EDC; Pierce): prepare immediately before use with sterile water.
  • 0.2% Tween‐20 (Sigma)
  • 1% sodium dodecyl sulfate (SDS; Sigma)
  • TE buffer ( appendix 2A)
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge with 1.5‐ml microtube rotor (e.g., Eppendorff 5415R)
  • 1‐ml transfer pipet, disposable plastic, pulled to a fine tip
  • Micropipettors (e.g., Brinkmann)
  • 0.5 M EDTA
  • 1.25 × 107/ml carboxylated microspheres (e.g., xMAP; Luminex)
  • Vortex mixer
  • Ultrasonic cleaner

Support Protocol 1: wLBMA Preparation and Quality Control

  Materials
  • Sterile water
  • 1 µg/µl target sequence (TS) oligonucleotide stocks: prepare in sterile water and store up to 1 month at 4°C or up to 1 year at −20°C (see Table 13.7.1 for complementary CPO sequences)
  • CBead sets ( protocol 1)
  • 1× and 1.5× tetramethylammonium chloride (TEMAC) buffers (see reciperecipes)
  • TE buffer ( appendix 2A)
  • 1 mg/ml streptavidin‐R‐phycoerythrin stock (Molecular Probes)
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Microcentrifuge with 1.5‐ml microcentrifuge tube rotor (e. g., Eppendorff 5415R)
  • Transfer pipets
  • Single and multichannel micropipettors (e.g., Brinkman)
  • 96‐well PCR plates (e.g., BioRad)
  • Thermal cycler
  • 96‐well flat‐bottom microtiter plates (e.g., Costar)
  • PCR plate‐sealing tape (e.g., BioRad)
  • Flow cytometer with temperature control and microtiter plate capability (e.g., Luminex 100; Luminex) with 532‐nm and 635‐nm excitation capability

Basic Protocol 2: PCR Synthesis of cDNA Using Random Hexamer Primers

  Materials
  • 100 mM dNTP stocks (e.g., Roche)
  • Sterile water
  • SuperScript III cDNA synthesis kit (Invitrogen)
    • 3 µg/ml random hexamer primer mix
    • 20× random hexamers
    • 5× dNTP
    • 5× 1st Str SuperScript III buffer
    • 0.1 M DTT
    • 40 U/µl RNaseOUT
    • 200 U/µl SuperScript III
  • 0.1 µg/µl RNA from patient or cell line: extract using Trizol (Invitrogen) according to the manufacturer's instructions
  • 1.5‐ml microcentrifuge tubes
  • Single and multichannel micropipettors (e.g., Brinkmann)
  • 96‐well PCR plates (e.g., BioRad)
  • Thermal cycler
  • Centrifuge with microtiter plate and PCR plate adapter (e.g., Eppendorff 5810R)
  • PCR plate‐sealing tape (e.g., BioRad)

Alternate Protocol 1: cDNA Synthesis Using Gene‐Specific Primers or Single‐Tube RT‐PCR

  Materials
  • 10× leukemia PPO mix (300 µM each primer): prepare by combining 10 µl of each 300 µM PPO primer stock (see Table 13.7.1 for primer design) and with sterile water to a final volume of 750 µl; store up to 1 week at 4°C or indefinitely at −20°C
  • Amplitaq Gold PCR kit (Applied Biosystems)
    • 10× PCR Gold Buffer II
    • 25 mM MgCl 2
    • AmpliTaq Gold DNA Polymerase
  • 100 mM dNTP stocks (e.g., Applied Biosystems)
  • PCR template ( protocol 3 or protocol 4) including positive (e.g., cell line K562) and negative controls (e.g., cell line SUDHL6)
  • Additional reagents and equipment for PCR (see protocol 3) and agarose gel electrophoresis (Voytas, ; optional)

Basic Protocol 3: Amplification of Target cDNA by Multiplex PCR

  Materials
  • Sterile water
  • 10× PCR Gold Buffer II (Applied Biosystems)
  • 20 U/µl Exonuclease I (New England Biolabs)
  • PCR template (Basic Protocol or protocol 4)
  • 300 µM biotin‐labeled PPO (see Table 13.7.1)
  • Amplitaq Gold PCR kit (Applied Biosystems)
    • 10× PCR Gold Buffer II
    • 25 mM MgCl 2
    • AmpliTaq Gold DNA Polymerase
    • 100 mM dNTP stocks (e.g., Applied Biosystems)
  • Additional reagents and equipment for PCR (see protocol 3)

Alternate Protocol 2: Exonuclease I‐Enabled ssPCR of Target cDNA

  Materials
  • TE buffer ( appendix 2A)
  • PCR samples (double‐ or single‐stranded ( protocol 5 or protocol 6) and positive (e.g., cell line K562) and negative (e.g., cell line SUDHL6) controls
  • wLBMA ( protocol 2)
  • 1 mg/ml streptavidin‐R‐phycoerythrin stock solution (Molecular Probes)
  • 1X TEMAC buffer (see recipe)
  • Single‐ and multichannel micropipettors (e.g., Brinkmann)
  • 96‐well PCR plates (e.g., BioRad)
  • PCR plate‐sealing tape (e.g., BioRad)
  • Thermal cycler
  • 96‐well flat‐bottom microtiter plates (e.g., Costar 3596)
  • Flow cytometer with temperature control and microtiter plate capability (e.g., Luminex 100; Luminex)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 13.7.1 Steps involved in the assay of gene fusions in leukemia using multiplex RT‐PCR and liquid bead arrays. (1) Conjugate capture probes to individual microsphere populations. (2) Mix multiple populations of conjugated microspheres to form a liquid bead microarray (LBMA). (3) Aliquot array into microtiter plate. (4) RNA extraction. (5) Reverse transcription. (6) Multiplex PCR. (6b) Asymmetric PCR. (7) Add PCR product to LBMA and hybridize. (8) Analyze microsphere fluorescence by flow cytometry.

Videos

Literature Cited

Literature Cited
   Bullinger, L., Dohner, K., Bair, E., Frohling, S., Schlenk, R.F., Tibshirani, R., Dohner, H., and Pollack, J.R. 2004. Use of gene‐expression profiling to identify prognostic subclasses in adult acute myeloid leukemia. New Engl. J. Med. 350:1605‐1616.
   Chamberlain, J.S., Gibbs, R.A., Ranier, J.E., Nguyen, P.N., and Caskey, C.T. 1988. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucl. Acids Res. 16:11141‐11156.
   Dorit, R.L. and Ohara, O. 1992. cDNA amplification using one‐sided (anchored) PCR. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 15.6.1‐15.6.10. John Wiley & Sons, Hoboken, N.J.
   Ferrando, A.A. and Look, A.T. 2000. Clinical implications of recurring chromosomal and associated molecular abnormalities in acute lymphoblastic leukemia. Semin. Hematol. 37:381‐395.
   Harris, N.L., Jaffe, E.S., Diebold, J., Flandrin, G., Muller‐Hermelink, H.K., Vardiman, J., Lister, T.A., and Bloomfield, C.D. 2000. The World Health Organization classification of neoplasms of the hematopoietic and lymphoid tissues: Report of the Clinical Advisory Committee meeting–Airlie House, Virginia, November, 1997. Hematol. J. 1:53‐66.
   Herling, M., Khoury, J.D., Washington, L.T., Du vic, M., Keating, M.J., and Jones, D. 2004. A systematic approach to diagnosis of mature T‐cell leukemias reveals heterogeneity among WHO categories. Blood 104:328‐335.
   Kern, W., Haferlach, T., Schoch, C., Sauerland, M.C., Heinecke, A., Wormann, B., Berdel, W., Buchner, T., and Hiddemann, W. 2004. Risk‐adapted therapy of AML: The AMLCG experience. Ann. Hematol. 83:S49‐51.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: standard procedures and optimization. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 15.1.1‐15.1.14. John Wiley & Sons, Hoboken, N.J.
   Linker, C.A. 1997. Risk‐adapted treatment of adult acute lymphoblastic leukemia (ALL). Leukemia 11:S24‐27.
   Pallisgaard, N., Hokland, P., Riishoj, D. C., Ped ersen, B., and Jorgensen, P. 1998. Multiplex reverse transcription‐polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia. Blood 92:574‐588.
   Pierce, K.E., Sanchez, J.A., Rice, J.E., and Wangh, L.J. 2005. Linear‐After‐The‐Exponential (LATE)‐PCR: Primer design criteria for high yields of specific single‐stranded DNA and improved real‐time detection. Proc. Natl. Acad. Sci. U.S.A. 102:8609‐8614.
   Ross, M.E., Mahfouz, R., Onciu, M., Liu, H.C., Zhou, X., Song, G., Shurtleff, S.A., Pounds, S., Cheng, C., Ma, J., Ribeiro, R.C., Rubnitz, J.E., Girtman, K., Williams, W.K., Raimondi, S.C., Liang, D.C., Shih, L.Y., Pui, C.H., and Downing, J.R. 2004. Gene expression profiling of pediatric acute myelogenous leukemia. Blood 104:3679‐3687.
   Smith, M., Arthur, D., Camitta, B., Carroll, A.J., Crist, W., Gaynon, P., Gelber, R., Heerema, N., Korn, E.L., Link, M., Murphy, S., Pui, C.H., Pullen, J., Reamon, G., Sallan, S.E., Sather, H., Shuster, J., Simon, R., Trigg, M., Tubergen, D., Uckun, F., and Ungerleider, R. 1996. Uniform approach to risk classification and treatment assignment for children with acute lymphoblastic leukemia. J. Clin. Oncol. 14:18‐24.
   Valk, P.J., Verhaak, R.G., Beijen, M.A., Erpelinck, C.A., Barjesteh van Waalwijk van Doorn‐Khosrovani, S., Boer, J.M., Beverloo, H.B., Moorhouse, M.J., van der Spek, P.J., Lowenberg, B., and Delwel, R. 2004. Prognostically useful gene‐expression profiles in acute myeloid leukemia. New Engl. J. Med. 350:1617‐1628.
   Voytas, D. 2000. Agarose gel electrophoresis. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.5A.1‐2.5A.9. John Wiley & Sons, Hoboken, N.J.
   Wallace, J., Zhou, Y., Usmani, G.N., Reardon, M., Newburger, P., Woda, B., and Pihan, G. 2003. BARCODE‐ALL: Accelerated and cost‐effective genetic risk stratification in acute leukemia using spectrally addressable liquid bead microarrays. Leukemia 17:1411‐1413.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library