RNAFISHonculturedcellsininterphase2

Post-labeling DNA processing and purification

1. Qiagen PCR clean up to get rid of unused oligonucleotides;

2. Add 20μl cot1DNA, 10μl ssDNA to compete for repetitive elements;

3. Precipitate the probe with 1/10 V 3 M NaOAc pH5.2, 2.5 V 100% ethanol at -80°C for 30 minutes;

4. Spin down pellet at 4°C for 20 minutes;

5. Wash pellet with 70% ethanol;

6. Resuspend pellet in 80μl hybridization mix.

The probe is now ready to be used. It can be stored at -20°C for at least a year.

Hybridization to RNA

1. Denature probe at 74°C for 10 minutes and let the probe anneal at 37°C for 30 minutes;

2. Meanwhile, dehydrate cells on slides through an ethanol series, 2 minutes each in 70%, 80%, 95%, 100% and then air dry;

3. Apply 5μl probe to the cells;

4. Place coverslip over probe and seal with rubber cement;

5. Incubate at 37°C overnight in a humidified chamber (light protected);

6. Float off coverslips with 4xSSC;.

7. Wash slides in the following sequence for 5 minutes at each step (unless otherwise stated) with gentle shaking:

1. 2xSSC, 50% formamide at 39°C for 3 times;

2. 2xSSC at 39°C for 3 times;

3. 1xSSC at room temperature once for 10 minutes;

4. 4xSSC at room temperature.

8. Counterstain cellular DNA with 5 microliter of 2.5mg/ml 4', 6-diamidino-2-phenylindole (DAPI) in 4xSSC, 0.1% TWEEN for 5 minutes at room temperature;

9. Wash with 4xSSC at room temperature for 5 minutes;

10. Mount coverslip with mounting media;

11. Seal the coverslip to the slide with nail polish;.

12. Slides may be visualized immediately or stored at 4°C.

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Materials & Reagents

1. Superfrost Plus slides - Sigma

2. Roboz slides - CellPoint Scientific, Gaithersburg, MD 20898-0757, order no: F107-HTC

3. 200mM vanadyl ribonucleoside complex (VRC, Invitrogen)

4. Paraformaldehyde extra pure - Merck 104005; 4% paraformaldehyde in PBS, pH 7.0 - made fresh from powder before each fixation.

5. Prime-IT ? II Random Primer Labeling kit - Strategene, 300385

6. Prime-It ? Fluor Fluorescence Labeling Kit - Strategene, 300380

7. Cy3-dCTP - Amersham Biosciences, PA53021, 25 nmol

8. Cy5-dCTP - Amersham Biosciences, PA55021, 25 nmol

9. Qiagen PCR Clean-up Kit

10. Cot1 DNA - Invitrogen

11. Salmon sperm DNA - Gibco

12. Formamide Fluka, 47470

13. Dextran sulphate - American Bioanlytical, Mg 500 000, ultra pure; to make 50% dextran sulphate in H₂0: mix at 4°C overnight.

14. BSA 10mg/ml NEB

15. 20xSSC stock: 3M NaCl, 0.3 M sodium citrate pH 7.4

16. Vectashield ? Mounting Medium - Vector Laboratories, H-1000

17. 4',6-diamidino-2-phenylindole (DAPI) - Molecular probes D-1306

18. 10mg made up as a 2.5mg/ml solution in methanol

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Figures

Figure 1. Xist RNA FISH in transgenic XXz mouse ES cells. The in situ hybridization is performed as described in the protocol. XXz cells express Xist RNA from 2 different loci. One expresses the full 17 kb Xist RNA, whilst the other site expresses a transgenic version, where the Xist 3' end is replaced by a LacZ sequence. Using DNA probes specific for the 3' end of Xist (Cy3, red) and LacZ (fluorescine, green), both Xist transcripts can be visualized simultanously in the cells. DNA is stained in blue (DAPI).