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.