96-well RNA In Situ Hybridization Protocol

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 5ul of 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, 5ul of restriction digest can be used directly.

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

3. Incubate at 37 degrees C for 2 hrs.

4. Add 10ul DNase I mix.

5. Incubate at 37 degrees C for 15 min.

6. Using multichannel pipettor, add 80ul 125 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 50ul 7.5M NH4 acetate to each well.

9. Transfer samples to 1.5ml eppendorf tubes containing 400ul 100% 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 50ul 50% 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 20ul embryos 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 200ul hybridization buffer

WITH dextran sulfate and 5ul of 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 100ul warm 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 200ul wash buffer to each well, then remove

wash buffer with LOW vacuum.

12. Repeat steps 10 and 11.

13. Add 200ul wash 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 200ul wash buffer, seal plate, then rock overnight at 55 degrees C.

17. Rinse embryos with 200ul PBT.

18. Add 200ul PBT, rock 30 min. at RT.

19. Remove PBT, add 200ul PBT + 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 200ul Alkaline Phosphatase buffer (AP buffer).

23. Wash 5 min at RT with AP buffer.

24. Add 200ul AP 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 200ul PBT.

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

27. Add 200ul 70% 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

287ul ddH2 0 100ul 10X Boehringer M. transcription buffer 50ul 10X dig-NTP mix 13ul RNase inhibitor (500U) 50ul T3 or T7 RNA polymerase (1000U) 500ul Total Volume

10X dig NTP mix

25.0ul 10mM digoxigenin UTP 4.5ul 100mM UTP 7.0ul 100mM CTP 7.0ul 100mM GTP 7.0ul 100mM ATP 19.5ul ddH2 O 70.0ul Total Volume

10X DNase I Buffer

0.2M Tris pH 8 0.1M MgCl2

DNase I mix

700ul ddH2 O 100ul 0.1M DTT 100ul 10X DNaseI Buffer 100ul DNase 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

Vendor order numbers

Boehringer Mannheim 1093274 anti-digoxigenin AP FAB fragments

Boehringer Mannheim 1383213 4-nitro blue tetrazolium chloride

Boehringer Mannheim 1383221 5-bromo-4-chloro-3-Indolyl-phosphate

Boehringer Mannheim 1209256 digoxigenin-11-UTP

Boehringer Mannheim 1277073 digoxigenin RNA labeling mix

Pharmacia 27-2025-01 NTP set 4X 25mMol

Pharmacia 27-0815-01 RNA guard Rnase inhibitor

Applied Sci. #AS 62049 lids for flexible vinyl 96-well plate (Falcon #3913)

Applied Sci. #AS 62046 U-bottom, flexible vinyl no lid 96-well plate (Falcon #3911)

Millipore MAVM 096-01 multi-screen filtration system vacuum manifold

Millipore MADV N65 10 DV, 0.65um Durapore; 96-well fitration plates