96-well RNA In Situ Hybridization Protocol

The RNA in situ procedure described below is based on the protocol developed by Tautz and Pfeifle (Chromosoma 98 (1989), p81), but adapted to allow the screening of 96 RNA probes on whole-mount Drosophila embryos at the same time.

Jasprien Noordermeer and Casey Kopczynski

I.PREPARATION OF EMBRYOS

1. Embryos were collected for 24 hours and washed 3X with 0.02% Triton X (Tx)

(15ml for up to 2ml embryos in a 50ml falcon tube).

2. Dechorionate 5 min. in 50% bleach.

3. Wash 3X with 0.02% Tx.

4. Fill tube with equal parts heptane and 4% paraformaldehyde/PBS (filtered).

5. Incubate 20 min. with frequent shaking.

6. Remove LOWER aqueous phase and replace with equal volume methanol.

7. Vortex at maximum speed for 1 min. then allow embryos to settle.

8. Remove UPPER phase along with embryos and vitelline membranes.

remaining at the interphase.

9. Rinse settled embryos 3X with methanol (Embryos can be stored at -20 degrees C at this point).

10. Rehydrate in 3:1 (MeOH: 4% paraformaldehyde/PBS) for 2 min., then 1:3 (MeOH: 4% paraformaldehyde/PBS) for 5 min.

11. Fix 10 min. in 4% paraformaldehyde/PBS.

12. Rinse 3X with PBS + 0.1% Tween 20 (PBT).

13. Wash embryos 3X with PBT before hybridization.

II. PREPARATION OF PROBE

The composition of the buffers used in sections II and III and general vendor information is described in section IV.

1. Place 5μlof linearized plasmid (100 - 500ng) into 96 well plate. (The CK cDNA miniprep DNA was linearized with Apa I). If restriction buffer is compatible with the polymerase buffer, 5μlof restriction digest can be used directly.

2. Using a multichannel pipettor, add 5μlof 2X polymerase mix.

3. Incubate at 37 degrees C for 2 hrs.

4. Add 10μlDNase I mix.

5. Incubate at 37 degrees C for 15 min.

6. Using multichannel pipettor, add 80μl125 mM NaCO₃ pH 10.2 to each well.

7. Incubate at 60 degrees C for 20 min. (This incubation time works best for 1-3 kb templates).

8. Place plate on ice, then quickly add 50μl7.5M NH₄ acetate to each well.

9. Transfer samples to 1.5ml eppendorf tubes containing 400μl100% EtOH. Vortex to mix.

10. Incubate at room temperature (RT) for 10 min.

11. Spin 13K for 15 min, drain well, then resuspend damp pellet in 50μl50% formamide / 50% TE pH 7.5 / 0.1% Tween 20.

12. Dilute probes to 25ug/ml where appropriate. This is a 50X stock for hybridization in 96 well plates.

III. HYBRIDIZATION IN 96 WELL PLATES

1. Prepare fixed embryos as described in section I.

2. Add 4 ml hybridization buffer WITHOUT dextran sulfate to 1.5ml of settled embryos.

3. Rock embryos for at least 1 hr at RT.

4. Using a multichannel pipettor with cut-off yellow tips, add 20μlembryos to each well of a 96 well filtration plate (Millipore MADV N65).

5. In a separate (non-filter) 96 well plate, mix in each well 200μlhybridization buffer

WITH dextran sulfate and 5μlof probe.

6. Using multichannel pipettor, add probes to wells of filtration plate.

7. Seal plate with tape and rock at 55 degrees C overnight.

8. Carefully remove cover, then add 100μlwarm wash buffer and place plate on vacuum manifold (Millipore MAVM 096 01).

9. Turn vacuum to LOWEST setting, press on top of plate to form seal. Once the last bit of hybridization buffer has been removed from wells quickly turn vacuum off.

(Make sure vacuum is set to lowest setting. If the vacuum is too high the embryos will become flattened and stick to the membrane.)

10. Empty the manifold tray containing hybridization buffer.

11. Use a multichannel pipettor to add 200μlwash buffer to each well, then remove

wash buffer with LOW vacuum.

12. Repeat steps 10 and 11.

13. Add 200μlwash buffer to each well, then rock 1hr. at 55 degrees C.

14. Remove wash under LOW vacuum.

15. Repeat steps 13 and 14 another 6 times.

16. Add 200μlwash buffer, seal plate, then rock overnight at 55 degrees C.

17. Rinse embryos with 200μlPBT.

18. Add 200μlPBT, rock 30 min. at RT.

19. Remove PBT, add 200μlPBT + 5% Normal Goat Serum + anti-digoxigenin Alkaline Phosphatase (1:2000 dilution), rock 2 hrs at RT.

20. Rinse twice with PBT .

21. Wash embryos 9 X for 10 min. in PBT.

22. Rinse twice with 200μlAlkaline Phosphatase buffer (AP buffer).

23. Wash 5 min at RT with AP buffer.

24. Add 200μlAP buffer containing Nitro Blue Tetrazolium (NBT) and Bromo-Chloro- Indolyl-Phosphatase (BCIP).

25. Incubate with rocking until desired color development is achieved (20 min. to overnight). To stop development of individual wells, remove staining solution and add 200μlPBT.

26. To stop entire plate, rinse plate 3X with PBT.

27. Add 200μl70% gycerol to each well.

28. Embryos are ready to mount or dissect once they have settled to the bottom of the well.

IV. BUFFER COMPOSITION AND VENDOR INFORMATION

2X Polymerase mix

287μlddH2 0

100μl10X Boehringer M. transcription buffer

50μl10X dig-NTP mix

13μlRNase inhibitor (500U)

50μlT3 or T7 RNA polymerase (1000U)

500μlTotal Volume

10X dig NTP mix

25.0μl10mM digoxigenin UTP

4.5μl100mM UTP

7.0μl100mM CTP

7.0μl100mM GTP

7.0μl100mM ATP

19.5μlddH₂ O

70.0μlTotal Volume

10X DNase I Buffer

0.2M Tris pH 8

0.1M MgCl₂

DNase I mix

700μlddH₂ O

100μl0.1M DTT

100μl10X DNaseI Buffer

100μlDNase I (100U, RNase-free)

1ml Total Volume

Probe Resuspension Buffer

5.0ml formamide

0.5ml 10X TE pH 7.4

4.4ml ddH₂ O

0.1ml 10% Tween

10ml total volume

Hybridization Buffer

150ml ultra pure redistilled formamide

60ml 20X SSC

6ml 50X Denhardts

7.5ml 10mg/ml tRNA

7.5ml 10mg/ml ssDNA (BOIL before adding)

0.3ml 50 mg/ml Heparin

3.0ml 10% Tween

****** ddH₂ O

270ml Total volume

******Add 30ml H₂ O to make hybridization buffer WITHOUT dextran sulfate.

******Add 30ml of 50% dextran sulfate to make hybridization buffer WITH dextran sulfate (5% d.s. final dilution).

Wash Buffer

250ml ultra pure redistilled formamide

50ml 20X SSC (autoclaved)

200ml ddH₂ O

2.5ml 20% Tween 20

500ml total volume

Alkaline PhosphataseBuffer (AP buffer)

100mM Tris pH9.5

100mM NaCl

50mM MgCl₂

0.1% Tween 20