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Method Article

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

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DOI:

10.3791/51628

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June 14th, 2014

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1Department of Reproduction and Development, Erasmus MC - University Medical Center

In This Article

Summary

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Abstract

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Introduction

Studying cells and tissues by means of fluorescent in situ hybridization (FISH) has, since its introduction in the late 1970s1, allowed researchers to study genes, chromatin organization and gene expression at the subcellular level. DNA FISH is frequently used in cytogenetics, karyotyping2, cancer diagnostics3 and pre-implantation genetic screening4, and has an important role in molecular research5-6 since it allows the detection of nucleic acids in their native environment. Single cell expression analysis by RNA FISH can detect native primary transcripts and noncoding RNAs being transcribed from chromosomes, and offers advantages to other techniques which assess gene expression at a population level, including for example quantitative RT-PCR, genome wide expression analysis or Northern blotting. By visualizing RNA transcripts originating directly from their sites of origin, it has for example been noticed that gene expression is stochastic7, and can sometimes be allele specific8. Improvements in the technique have even allowed the detection and quantification of single mRNA molecules within cells9-11.

The basic principle of FISH consists of hybridization of nucleic acids within the cell to a nucleic acid probe by means of highly specific Watson and Crick base-pairing. The probe can be either directly or indirectly detected, resulting in a signal which can be microscopically visualized. Initial attempts consisted of radioactively labeled probes, which had drawbacks based on safety issues, limited spatial resolution and the ability to detect only one target at a time12-14. The subsequent development of nonradioactive labels, including fluorochromes, haptens, and enzymes, has allowed the wide spread use of FISH as a routine molecular biology technique. The FISH probe can either be directly labeled with fluorochromes by chemical linking of fluorescent molecules to nucleic acid sequences15 and integration of fluorescently labeled nucleotides16-19, or the probe can be indirectly visualized after integration of haptens (including biotin and digoxigenin) and immunological detection of haptens by hapten specific antibodies conjugated to fluorescent reporter molecules20-21. The latter approach allows signal amplification by using several layers of fluorescently labeled antibodies which are used to enhance the original signal, and enables the detection of RNA species which are expressed at low levels. By combining direct and indirect labeling techniques and various haptens, several targets can be simultaneously visualized within the same cell.

One of the most important steps in FISH protocols is the hybridization of the probe to its target. Although in theory, a probe will specifically bind only to its target, in practice, this specificity is not always achieved, as probes may bind to homologous regions, and hybridization conditions will not always be ideal for a certain DNA region or RNA species. Post-hybridization washes are therefore of particular importance, as they can increase the stringency of the FISH procedure, and can prevent nonspecific binding of FISH probes, which would result in a high level of background noise. As RNA molecules are single stranded they can easily be hybridized to a FISH probe. In contrast, the double stranded DNA molecule first needs a denaturation step, after which a probe can hybridize. This is usually achieved by heating of the specimen, which results in denaturation of the DNA. However, under these harsh conditions, fragile, single stranded RNA molecules might be lost. Therefore, combined DNA-RNA FISH requires significant optimization of the conditions, and is more technically challenging compared to the separate detection of only RNA or DNA.

Here we present a detailed protocol of combined, simultaneous DNA-RNA FISH which has allowed us to study X chromosome inactivation (XCI) in differentiating female mouse embryonic stem cells22-24. XCI is a crucial epigenetic mechanism for female embryonic development25, and results in heterochromatinization and hence silencing of one of the two X chromosomes in female individuals26-27. Essential to this process is the noncoding RNA Xist28-30, which is regulated by the RNF1222-23 and REX124 proteins. Xist expression becomes upregulated on the future inactive X chromosome (Xi) during embryonic development or upon ES cell differentiation in vitro, and can spread along the X chromosome and thereby attract chromatin remodeling enzymes which result in the transcriptional shutdown of the X chromosome31. This spreading of Xist RNA can be visualized by RNA FISH as a coating of the X chromosome, which is also referred to as an Xist cloud. Since female ES cells can lose one of their X chromosomes due to genomic instability, we and others have employed combined DNA-RNA FISH to study XCI, to make sure that only karyotypically stable cells are assessed in the analysis of this important process22,32-34. As with every molecular biology technique, several different excellent protocols have been published35-38. Here we present our method, starting from the induction of differentiation in mouse ES cells, fixation of cells, labeling of FISH probes by Nick-translation, pre-treatment of fixed cells to allow permeabilization and subsequent probe uptake, hybridization of the probe to the target, and finally detection of the probe by fluorescently labeled antibodies. The herein presented protocol allows the faithful detection of Xist RNA and the X chromosome within a period of two days, and the basics of this technique can likely be adapted to other systems and areas of research.

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Protocol

1. Induction of Differentiation in Female Embryonic Stem Cells to Induce X Chromosome Inactivation

NOTE: Female mouse embryonic stem cells (available upon request) are grown under standard ES cell conditions on gelatinized culture dishes coated with mouse embryonic fibroblasts (MEFs). We here assume that the reader is familiar with standard cell culture techniques39-41. To induce differentiation, ES cells grown in T25 dishes will be separated from the MEFs, and will be plated in differentiation medium.

  1. Remove ES medium from the ES cell culture, and wash twice with cell culture PBS.
  2. Add 1.5 ml trypsin-EDTA (pre-warmed to 37 °C), and incubate cells at 37 °C for 7 min. After 3.5 min, shake the dish gently to break up the colonies.
  3. Inactivate the trypsin-EDTA by adding 3.5 ml of differentiation medium to the cells. Pipette cells up and down to obtain a single cell solution, and collect in a 15 ml tube. Spin for 5 min at 200 x g, and collect cells in 10 ml of differentiation medium.
  4. Add cell suspension to a non-gelatinized culture dish, and incubate for 1 hr in a cell culture incubator. NOTE: MEFs will be able to adhere to the non-gelatinized culture dish, whereas ES cells will remain in suspension.
  5. In the meantime, add sterilized glass coverslips (24 x 24 mm) to 6-well plates, add 0.2% gelatin, and incubate for minimal 5 min at room temperature.
  6. After 1 hr, collect the supernatant of the pre-plated ES cell culture, which will mainly consist of non-adhering ES cells. Plate ES cells to 6-well plates containing coverslips. NOTE: Use ⅙th of a confluent T25 dish for 6 wells of a 6-well plate. This will allow a confluent differentiation well after 3 days of differentiation. Change medium on a daily basis. For longer differentiation time courses, plate pre-plated ES cells in differentiation medium in bacterial dishes, and incubate on a cell shaker integrated into the cell culture incubator. This will allow the cells to form embryoid bodies, which can be plated to coverslips 1-2 days prior to fixation.

2. Fixation of Differentiated ES Cells for Subsequent DNA-RNA FISH Analysis

  1. Remove differentiation medium from differentiation cultures, and wash twice with cell culture PBS. NOTE: Add PBS carefully to prevent the cells being washed away.
  2. Remove cell culture PBS and fix cells by adding 4% paraformaldehyde (PFA)/PBS, and incubate for 10 min at room temperature. CAUTION: PFA is toxic and can cause significant health risk when being inhaled or when in contact to skin or eyes. Furthermore, PFA is carcinogenic.
  3. Remove 4% PFA/PBS, and wash coverslips 3x with 70% ethanol. NOTE: Correct washing is important, as any trace of PFA will interfere with subsequent FISH steps. Coverslips can be stored in 70% ethanol at -20 °C for several months prior to FISH analysis.

3. Labeling of DNA Probes for FISH Experiments

NOTE: Obtain a DNA fragment of the gene of interest (minimal length >2 kb for RNA FISH, or >20 kb for DNA FISH), or a BAC covering the gene of interest. For RNA detection, a smaller probe can be sufficient compared to DNA detection, since most likely transcription will result in multiple transcripts, which can be simultaneously detected. For a strong signal on the single copy DNA strand, a longer probe is required, to be able to detect a sufficient number of incorporated haptens. Preferably, a non-transcribed genomic region is chosen for designing the DNA probe. In addition, when using a smaller probe to detect the RNA, it will be prevented that the genomic loci from which the RNA is transcribed is being detected in the combined DNA-RNA FISH approach, although this cannot be fully excluded. To detect mouse Xist, a 5.5 kb cDNA probe was used. For detection of the X chromosome, several BAC probes can be used. Good results have been obtained with a mixture of BAC RP23-100E1 and BAC CT7-474E4, which are located in close proximity to the Xist locus. Depending on the gene or chromosome of interest, several different probes might be required to obtain optimal results. Whenever working with materials which will be used for RNA-FISH, use sterile filter tips, RNAse free H2O, and work in a clean environment.

  1. Take 1 µg of DNA and dilute in H2O in a total volume of 16 µl. Add 4 µl of digoxigenin or biotin labeling solution (see reagents), mix by vortexing and incubate at 16 °C for 90 min. NOTE: The biotin or digoxigenin-labeled nucleotides will now be incorporated by nick-translation.
  2. In the meantime, prepare G50 columns to remove non-integrated nucleotides. Take a 2 ml syringe, remove the stamper, and add sterilized cotton into the syringe. Add G50 balls in solution to fill the syringe, place syringe in a 15 ml tube and centrifuge at 642 x g for 1 min. NOTE: Around 80% of the syringe should now be filled with G50. Repeat when less G50 is present.
  3. After 90 min, add 40 µl of H2O to the nick-translation reaction mixture, and add the reaction to the G50 column. Add an empty tube under the column, and centrifuge at 642 x g for 2 min. Collect the flow through in a reaction tube.
  4. Measure the volume of the flow through with a pipette, and add H2O to reach a total volume of 200 µl. Precipitate the flow through by adding 13.3 µl salmon sperm DNA (10 mg/ml), 13.3 µl yeast tRNA (10 mg/ml), 26.7 µl mouse Cot-1 DNA (1 mg/ml) and 28 µl 2 M NaAc, pH 5.6. Mix by vortexing, and add 533 µl ice cold 100% ethanol. Shake the tube, and centrifuge at 16,200 x g for 30 min at 4 °C.
  5. Remove the supernatant, and wash pellet twice with 70% ethanol. Centrifuge at 16,200 x g for 5 min at 4 °C, and air dry the pellet. Add 50 µl of 50+ hybridization solution, and incubate at 37 °C for 30 min to facilitate dissolving. NOTE: Labeled probe can be stored for several years at -20 °C. CAUTION: 50+ hybridization solution contains formamide, which is toxic, can irritate skin, eyes and the respiratory system, and is also a teratogen.

4. Permeabilization, Pre-treatment and Hybridization of Fixed Cells for Combined DNA-RNA FISH

  1. Rehydrate fixed cells on coverslips, by removing 70% ethanol and the addition of cell culture PBS to the 6-well plates. Incubate for 5 min. Repeat in total three times.
  2. Remove cell culture PBS, and add 0.2% pepsin to the 6-well plates, to permeabilize the cells. Incubate 6-well plates in a water bath at 37 °C for 4 min. Remove pepsin, and inactivate with RNAse free H2O.
  3. Post-fix cells by incubating with 4% PFA/PBS for 5 min at room temperature. After fixation, wash twice with cell culture PBS for 5 min.
  4. Dehydrate cells by incubating with 70% ethanol for 3 min, 90% ethanol for 3 min and 100% ethanol for 3 min. Transfer coverslips out of 6-well plates, and air dry coverslips for several minutes.
  5. Age cells by incubating coverslips at 65 °C for 1 hr on a heat plate.
  6. Pre-hybridize FISH probe with mouse Cot-1 DNA by adding together for every coverslip to be analyzed, 25 µl of 50+ hybridization solution, 0.5 µl mouse Cot-1 DNA, 1 µl of biotin-labeled Xist probe and 1 µl of digoxigenin labeled BAC probe detecting the X chromosome. Mix by vortexing, and denature at 99 °C for 5 min. Incubate immediately at 37 °C for 45 min, to allow Cot-1 DNA to hybridize to repeat sequences present in the probes.
  7. After aging, spot 50 µl of denaturation buffer (consisting of 70% formamide/2x SSC/10 mM phosphate buffer) on a glass slide, and put coverslips on top with cells facing towards the denaturation buffer. Incubate at 65 °C for 2 min.
  8. Add coverslips back to 6-well plates, and post-fix cells by incubating in ice cold 70% ethanol for 5 min.
  9. Dehydrate cells by subsequent incubation in 70% ethanol for 3 min, 90% ethanol for 3 min and 100% ethanol for 3 min. Remove coverslips from 6-well plates, and let air dry for several minutes.
  10. Spot pre-hybridized probe on a glass slide, and incubate coverslip on top. Place slides in a humidified chamber filled with 50 ml 50% formamide/2x SSC, and incubate overnight at 37 °C.

5. Post-hybridization Washes and Antibody Mediated Detection of Probe

  1. Fill 6-well plates with 2x SSC, and pre-warm till 42 °C. Add coverslips after overnight incubation, and incubate at 42 °C for 5 min.
  2. Remove 2x SSC, and incubate coverslips with 50% formamide/2x SSC for 10 min at 42 °C. Repeat in total 3 times (30 min of washing altogether).
  3. Remove 50% formamide/2x SSC, and wash slides with Tris-saline-Tween (TST) for 2 x 5 min at room temperature.
  4. Spot 50 µl Tris-saline-BSA (TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for blocking during 30 min at room temperature in a TST-humidified dark chamber.
  5. Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.
  6. Spot 50 µl of sheep-anti-dig antibody (1:500 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside-down for the first incubation step during 30 min at room temperature in a TST-humidified dark chamber.
  7. Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.
  8. Spot 50 µl of rabbit-anti-sheep antibody conjugated with FITC (1:250 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the second incubation step during 30 min at room temperature in a TST-humidified dark chamber.
  9. Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.
  10. Spot 50 µl of goat-anti-rabbit antibody conjugated with FITC (1:250 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the third incubation step during 30 min at room temperature in a TST-humidified dark chamber.
  11. Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.
  12. Spot 50 µl of mouse-anti-biotin antibody (1:200 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the fourth incubation step during 30 min at room temperature in a TST-humidified dark chamber.
  13. Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.
  14. Spot 50 µl of donkey-anti-mouse antibody conjugated with Rhodamine (1:250 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the fifth incubation step during 30 min at room temperature in a TST-humidified dark chamber.
  15. Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.
  16. Spot 50 µl of goat-anti-horse antibody conjugated with Rhodamine (1:250 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the sixth incubation step during 30 min at room temperature in a TST-humidified dark chamber. NOTE: the goat-anti-horse antibody will recognize the donkey-anti-mouse antibody; when available to the researcher, a goat-anti-donkey antibody will work as well.
  17. Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST. Wash additional 5 min with Tris-saline (TS).
  18. Dehydrate cells by subsequent incubation in 70% ethanol for 3 min, 90% ethanol for 3 min and 100% ethanol for 3 min. Remove coverslips from 6-well plates, and let air dry for several minutes.
  19. Spot a drop of mounting medium with DAPI (see reagents) on a glass slide, and add coverslip upside down on top. Seal slides with nail varnish and assess FISH results by fluorescent microscopy (Figure 1).

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Results

Using the above mentioned protocol for combined DNA-RNA FISH, we have been able to visualize X chromosome inactivation in differentiating female embryonic stem cells. Figure 1 shows a representative example of an DNA-RNA FISH experiment, where we detected both Xist (which is visible as an so-called Xist RNA cloud on the inactive X chromosome and a basal transcription pinpoint on the active X chromosome), and a region of the X chromosome, which is visible as a pinpoint signal. Note that ...

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Discussion

Combined DNA-RNA FISH can be technically challenging, as conditions suitable for DNA FISH can be too harsh for less stable RNA molecules. Several approaches have been applied to study both DNA and RNA in the same cell, circumventing the need for simultaneous incubation with probes detecting both the DNA and RNA. For example, in a superimposition approach, first RNA FISH is performed, and cells are imaged, and coordinates are taken. Subsequently, the same slides are used for DNA FISH, during which the signal of RNA FISH i...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors thank all previous and present members of the department of Reproduction and Development of the Erasmus MC for helpful and stimulating discussions during the past years. We also want to thank the three anonymous reviewers for providing helpful feedback. Work in the Gribnau lab is supported by funding from the Dutch research council (NWO-VICI) and an ERC starting grant.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
female mouse embryonic stem cellswill be made available upon request
mouse embryonic fibroblastscan be derived from E13.5 embryos, or via commercial sources e.g. Millipore
ES cell culture mediumDMEM, 15% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, non-essential amino acids, 0.1 mM γ-mercaptoethanol, and 1,000 U/ml LIF. Filter sterilize and store at 4 °C for maximum of 2 weeks.
LIFChemiconESG1107
DMEMInvitrogen11960085
Fetal Calf SerumInvitrogen10099141
Penicillin–streptomycin (100x)Invitrogen15140-122
Non-essential aminoacidsInvitrogen11140-050
γ-mercaptoethanolSigma-AldrichM7522
ES cell differentiation mediumIMDM-Glutamax, 15% heat inactivated fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, non-essential amino acids, 37.8 μl/L monothioglycerol and 50 μg/ml ascorbic acid
IMDM-GlutamaxInvitrogen31980-030
Ascorbic acid (50 mg/ml)Sigma-AldrichA4403
monothioglycerolSigma-AldrichM6145
T25 cell culture dishesGreiner Bio-one690160
6-well cell culture dishesGreiner Bio-one662160
cell culture grade PBSSigma-AldrichD8537
Trypsin-EDTA 0.25% (v/v) trypsin/0.2% (w/v) EDTA in PBSGibco25200-056
Falcon tubesFalcon352196
24 x 24 mm coverslipsMenzle780882
gelatinSigma-AldrichG1890-100Gprepare a 0.2% gelatin/cell culture PBS solution, and autoclave
PepsinSigma-AldrichP7000Dilute to 0.2% in 0.01 M HCl (pH 2.0)
absolute 100% ethanolSigma-Aldrich32221-2.5Luse absolute 100% ethanol and RNAse free water to make 70% and 90% ethanol solutions
RNAse free waterBaxterTKF7114
sterile filter tipsRainin Bio CleanGP-L10F, GP-L200F, RT-1000F
digoxigenin-labeling kit (DIG-Nick translation)Roche11745816910
biotin-labeling kit (Biotin Nick translation)Roche11745824910
Sephadex G50Sigma-AldrichG5080
2 ml syringeBD Plastipak300013
sterilized cotton
Eppendorf tubesEppendorf30120086
yeast tRNA 10 mg/mlInvitrogen15401-029
Salmon Sperm DNA 10 mg/mlInvitrogen15632-011
mouse cot-1 DNA 1 mg/mlInvitrogen18440-016
Eppendorf bench top microcentrifugeEppendorfModel 5417R
Eppendorf refrigerated centrifugeEppendorfModel 5810R
2 M NaAc, pH 5.6Sigma-AldrichS7670
50+ hybridization solution50% formamide, 2x SSC, 50 mM phosphate buffer pH 7, 10% dextran sulfate pH 7
formamideAcros organics3272350000
dextran sulfateFluka31403
10 mM phosphate buffer, pH 7add 57.7 ml of 1 M Na2HPO4 and 42.3 ml 1 M NaH2PO4; use DEPC treated water and autoclave
denaturation buffer70% formamide/2x SSC/10 mM phosphate buffer pH 7
Tween-20Sigma-AldrichP2287
2 M Tris pH 7.5 solution
Tris-saline-Tween washing solution (TST)100 ml 10x saline, 50 ml 2M Tris, 500 μl Tween-20, add RNAse free water till 1 L
10x saline solutiondissolve 85 g NaCl in 1 L RNAse free H2O, and autoclave
Tris-saline-BSA (TSBSA)500 μl 10x saline, 250 μl 2 M Tris, 1 ml 100x BSA, 3.25 ml RNAse free H2O
BSA, purified, 100xNew England BiolabsB9001S
sheep-anti-dig antibodyRoche diagnosticsuse 1:500 diluted in TSBSA
rabbit-anti-sheep antibody conjugated with FITCJackson labsuse 1:250 diluted in TSBSA
goat-anti-rabbit antibody conjugated with FITCJackson labsuse 1:250 diluted in TSBSA
mouse-anti-biotin antibodyRoche diagnosticsuse 1:200 diluted in TSBSA
donkey-anti-mouse antibody conjugated with RhodamineJackson labsuse 1:250 diluted in TSBSA
goat-anti-horse antibody conjugated with RhodamineJackson labsuse 1:250 diluted in TSBSA
Tris-saline washing solution (TS)10 ml 10x saline, 5 ml 2 M Tris, 85 ml RNAse free H2O
Vectashield mounting medium with DAPIVectorlabsH-1200
nail varnish
fluorescent miscroscope

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Combined DNA RNA FISHXist RNA DetectionDNA Probe LabelingNick TranslationImmunofluorescent MicroscopyParaformaldehyde FixationHybridization BufferFluorescent Antibodies
Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells
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