A Multifaceted FISH Approach to Study Endogenous RNAs and DNAs in Native Nuclear and Cell Structures

Meg Byron1, Lisa L. Hall1, Jeanne B. Lawrence1

1 Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 4.15
DOI:  10.1002/0471142905.hg0415s76
Online Posting Date:  January, 2013
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Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre‐mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results. Curr. Protoc. Hum. Genet. 76:4.15.1‐4.15.21. © 2013 by John Wiley & Sons, Inc.

Keywords: FISH; fluorescence in situ hybridization; nuclear structure; DNA; RNA; immunofluorescence; cytogenetics; histochemistry; chromosomes

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Cells for Fluorescence In Situ Hybridization
  • Basic Protocol 2: Preparation of Probes for In Situ Hybridization by Nick Translation
  • Basic Protocol 3: Hybridization to RNA
  • Alternate Protocol 1: Hybridization to DNA
  • Alternate Protocol 2: RNA or DNA Hybridization with Oligonucleotide Probes
  • Alternate Protocol 3: Hybridization to RNA and DNA Simultaneously in Two Distinct Colors
  • Alternate Protocol 4: Protein Detection with Hybridization to RNA or DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Cells for Fluorescence In Situ Hybridization

  • Monolayer cells grown on 22 × 22−mm glass coverslips (non‐adherent cells can be attached using Cell‐Tak from BD Bioscience)
  • Hanks' balanced salt solution (HBSS; appendix 2D)
  • Cytoskeletal buffer (CSK)
  • 10% Triton X‐100 (Roche)
  • 200 mM vanadyl ribonucleoside complex (VRC; Sigma; dispense 500‐µl aliquots on ice and store up to several months at −20°C)
  • 4% (v/v) paraformaldehyde (PFA; Ted Pella) in 1× PBS
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • 70% (v/v) ethanol
  • Coplin jars for 22 × 22−mm coverslips (Thomas Scientific)
  • Fine forceps

Basic Protocol 2: Preparation of Probes for In Situ Hybridization by Nick Translation

  • Nucleotide mix: 600 µM each dATP, dCTP, and dGTP (Roche)
  • Clean DNA
  • 50 nmol biotin‐16‐dUTP or 25 nmol digoxigenin‐11‐dUTP (Roche)
  • Nuclease‐free water
  • Nick translation mix (Roche)
  • 0.5 M EDTA ( appendix 2D)
  • 5% (w/v) sodium dodecyl sulfate (SDS; appendix 2D)
  • ssDNA/tRNA solution (see recipe)
  • 3 M sodium acetate ( appendix 2D)
  • 70% and 95% (v/v) ethanol, 4°C or colder
  • Microcentrifuge tubes
  • 16°C water bath
  • 80°C heating block

Basic Protocol 3: Hybridization to RNA

  • Coverslips with fixed, extracted cells (see protocol 1)
  • 100% ethanol, 4°C or colder
  • 10 ng/µl biotin‐ or digoxigenin‐labeled DNA probe (see protocol 2)
  • Human Cot‐1 DNA (Roche) or mouse Cot‐1 DNA (Invitrogen)
  • ssDNA/tRNA solution (see recipe)
  • Formamide (Sigma)
  • Hybridization buffer (see recipe)
  • RNase inhibitor (RNasin, Promega, or RNase OUT, Invitrogen)
  • Nail polish (clear)
  • 4×, 2×, and 1× SSC (see recipe)
  • Dylight 488− or 594−labeled streptavidin (Jackson Immuno Research) or fluorescein‐conjugated anti‐digoxigenin antibody (Roche)
  • 1% (w/v) bovine serum albumin (BSA) in 4× SSC
  • 0.1% (v/v) Triton X‐100 (Roche) in 4× SSC
  • 100 µg/ml DAPI (Sigma) in 1× PBS (store up to several months in the dark at 4°C)
  • 1× phosphate‐buffered saline (PBS; see recipe)
  • Vectashield anti‐fade medium (Vector Labs)
  • 80°C heating block
  • Parafilm
  • Glass microscope slides (Corning)
  • Forceps
  • Shaker
  • Slide folders

Alternate Protocol 1: Hybridization to DNA

  • 70% (v/v) ethanol, 4°C or colder
  • 70% (v/v) formamide (Sigma) in 2× SSC (see recipe), freshly prepared
  • 80°C water bath
  • Coplin jar prewarmed to 80°C in water bath
  • 10‐20 ml beaker
  • Microwave oven
  • Thermometer

Alternate Protocol 2: RNA or DNA Hybridization with Oligonucleotide Probes

  • Primary antibody of interest
  • Fluorescently conjugated secondary antibody
  • 1% (w/v) bovine serum albumin (BSA) in 1× PBS
  • 0.1% (v/v) Triton X‐100 (Roche) in 1× PBS
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Literature Cited

Literature Cited
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   Clemson, C.M., McNeil, J.A., Willard, H.F., and Lawrence, J.B. 1996. XIST RNA paints the inactive X chromosome at interphase: Evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132:259‐275.
   Clemson, C.M., Hall, L.L., Byron, M., McNeil, J., and Lawrence, J.B. 2006. The X chromosome is organized into a gene‐rich outer rim and an internal core containing silenced nongenic sequences. Proc. Natl. Acad. Sci. U.S.A. 103:7688‐7693.
   Clemson, C.M., Hutchinson, J.N., Sara, S.A., Ensminger, A.W., Fox, A.H., Chess, A., and Lawrence, J.B. 2009. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol. Cell 33:717‐726.
   Hall, L.L., Byron, M., Sakai, K., Carrel, L., Willard, H.F., and Lawrence, J.B. 2002. An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT‐1080 cells. Proc. Natl. Acad. Sci. U.S.A. 99:8677‐8682.
   Hall, L.L., Smith, K.P., Byron, M., and Lawrence, J.B. 2006. Molecular anatomy of a speckle. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 288:664‐675.
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   Langer, P.R., Waldrop, A.A., and Ward, D.C. 1981. Enzymatic synthesis of biotin‐labeled polynucleotides: Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. U.S.A. 78:6633‐6637.
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   Lawrence, J.B. and Singer, R.H. 1985. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res. 13:1777‐1799.
   Lawrence, J.B. and Clemson, C.M. 2008. Gene associations: True romance or chance meeting in a nuclear neighborhood. J. Cell Biol. 182:1035‐1038.
   Lawrence, J.B., Villnave, C.A., and Singer, R.H. 1988. Sensitive, high‐resolution chromatin and chromosome mapping in situ: Presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell 52:51‐61.
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   Lichter, P., Bray, P., Ried, T., Dawid, I.B., and Ward, D.C. 1992. Clustering of C2‐H2 zinc finger motif sequences within telomeric and fragile site regions of human chromosomes. Genomics 3:999‐1007.
   Pardue, M.L. and Gall, J.D. 1969. Molecular hybridization of radioactive DNA to the DNA of cytological preperations. Proc. Natl. Acad. Sci. U.S.A. 64:600‐604.
   Pinkel, D., Landegent, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J., and Gray, J. 1988. Fluorescence in situ hybridization with human‐specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. U.S.A. 85:9138‐9142.
   Rossner, M. and Yamada, K.M. 2004. What's in a picture? The temptation of image manipulation. J. Cell Biol. 166:11‐15.
   Smith, K.P. and Lawrence, J.B. 2000. Interactions of U2 gene loci and their nuclear transcripts with Cajal (coiled) bodies: Evidence for PreU2 within Cajal bodies. Mol. Biol. Cell 11:2987‐2998.
   Smith, K.P., Moen, P.T., Wydner, K.L., Coleman, J.R., and Lawrence, J.B. 1999. Processing of endogenous pre‐mRNAs in association with SC‐35 domains is gene specific. J. Cell Biol. 144:617‐629.
   Smith, K.P., Byron, M., Johnson, C., Xing, Y., and Lawrence, J.B. 2007. Defining early steps in mRNA transport: Mutant mRNA in myotonic dystrophy type I is blocked at entry into SC‐35 domains. J. Cell Biol. 178:951‐964.
   Tam, R., Shopland, L.S., Johnson, C.V., McNeil, J.A., and Lawrence, J.B. 2002. Applications of RNA FISH for visualizing gene expression and nuclear architecture. In FISH: A Practical Approach (B.G. Beatty, S. Mai, and J. Squire, eds.) pp. 93‐118. Oxford University Press, New York.
   Tam, R., Smith, K.P., and Lawrence, J.B. 2004. The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin unlike most human telomeres. J. Cell Biol. 167:269‐279.
   Xing, Y.G. and Lawrence, J.B. 1991. Preservation of specific RNA distribution within the chromatin‐depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation. J. Cell Biol. 112:1055‐1063.
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