RNA In Situ Hybridization with Drosophila Embryos

Benjamin Pease, revised August 2008

benjamin_pease@hms.harvard.edu

 

Adapted from

Lehmann R, Tautz D. 1994. In situ hybridization to RNA. Methods Cell Biol. 44:575-98
Nagaso H, Murata T, Day N, Yokoyama KK. 2001. Simultaneous detection of RNA and protein by in situ hybridization and immunological staining. J Histochem Cytochem. 49:1177-82.

The procedure below describes the method used for detecting strand-specific RNA transcripts in Drosophila embryos. Single-stranded digoxigenin-labeled RNA probe is synthesized from a cloned genomic template. The probe is then hybridized to fixed embryos, and detected using a DIG antibody coupled to alkaline phosphatase, which stains purple in the presence of NBT and X-Phosphate. The in situ procedure itself takes 2 days.

 

Reagents Needed

T7 RNA Polymerase (NEB)
10x DIG Labeling Mix: 10 uM ATP, 10 uM CTP, 10 uM GTP, 6.5 uM UTP, 3.5uM DIG-UTP (Roche DIG Northern Starter Kit)
RNase Inhibitor (Promega)
anti-DIG-AP: fab fragments from sheep (Roche DIG Northern Starter Kit)
Xylene

Stocks (store @-20^oC)

NBT
75mg/mL nitroblue tetrazolium in 70% dimethylformamide (DMF)

X-Phosphate
50mg/mL 5-bromo-4-chloro-3-indolyl phosphate in DMF

Buffers (DEPC treated)

PBT
130mM NaCl
7mM Na₂HPO₄
3mM KH₂PO₄
0.1% Tween 20
pH 7.2

Blocking Solution (Roche DIG Northern Starter Kit)

Hybridization Buffer (HB-A)
50% deionized formamide
5x SSC
100 ug/mL tRNA
50 ug/mL heparin
0.1% Tween 20

Hybridization Wash Buffer (HB-B)
50% deionized formamide
5x SSC

Maleic Acid Wash Buffer (MAB)
0.1M maleic acid
0.15M NaCl
0.1% Tween 20
pH 7.5

AMP Staining Buffer
1% 2-amino-2-methyl-1-propanol
100mM NaCl
50mM MgCl2
pH 9.2

DIG  Probe Synthesis

1. Set up RNA probe synthesis reaction as follows:

2 uL 10x RNA Pol Buffer (NEB)
2 uL 10x DIG Labeling Mix (10 uM ATP, 10 uM CTP, 10 uM GTP, 6.5 uM UTP, 3.5uM DIG-UTP)
1 ug linearized DNA in vector containing T7 promoter (longer template will increase signal)
50 U RNA Polymerase (T7 or T3)
40 U RNase Inhibitor
ddH₂O to 20 uL total volume
Leave at 42^oC for 90 minutes
After 90 minutes, ad 2 uL 0.2M EDTA to stop reaction

2. Check RNA probe by loading 2uL newly synthesized RNA probe onto EtBr agarose gel alongside DNA and RNA standards. It is important that the gel box is clean and gel buffer is fresh to minimize RNase contamination. There should be two bands -- one for the linearized DNA, and the other for the probe. If the RNA band is smeared from degradation, it's ok because the probe is fragmented in the next step.

 

Fragment and store the probe

1. Add one volume (20uL) of DEPC-treated 0.2M sodium carbonate to remaining RNA probe and leave at 60^oC for 10 minutes.
2. Place on ice, add one volume (40uL) 5M ammonium acetate.
3. Ethanol precipitate. Use co-precipitant to visualize pellet. Resuspend in 100uL hybridization buffer. Store at -80^oC.

 

Probe Concentration

Note: The exact probe concentration is not critical. However, this assay can be a useful troubleshooting tool.

1. Add 0.2 uL probe to 9.8uL Dilution Buffer.
2. From this, make a 1:10 dilution series in Dilution Buffer (5 tubes). Do the same for actin probe standard (Roche DIG Northern Starter Kit).
3. Pipette 1uL from each dilution onto nylon membrane alongside DIG-RNA standard and UV crosslink (~1200 uJoules/cm² or ~30 seconds).
4. Wash the membrane in maleic acid wash buffer (MAB) for 2 min.
5. Incubate for 30 minutes in Blocking Solution.
6. Incubate 30 minutes in Antibody Solution (anti-DIG-AP).
7. Wash  2 x 15 minutes in MAB
8. Incubate 5 minutes in AMP Staining Buffer.
9. Stain in AMP Staining Buffer + 4.5 uL/mL NBT + 3.5 uL/mL X-Phosphate. Purple spots should appear within 5 minutes. Continue staining for 30 minutes.
10. Wash in PBT. Compare intensity of dilution series to actin probe standard

 

Embryo Fixation

1. Collect embryos after 24 hours. The embryos will be at various stages of development. from 0 to 24 hours.
2. Using suction filtration, wash the embryos with ddH₂O.
3. Dechorionate by immersing the embryos in freshly prepared 50% bleach solution for 2 minutes. Mix with a Pasteur pipet.
4. Rinse with ddH₂O.
5. In a glass test tube, Immerse embryos in 1:9 37% formaldehyde:Fixation Buffer (~4% formaldehyde final). Add an equal volume of heptane. The bottom phase will consist of the 4%formaldehyde buffer. The top phase will be heptane. Most of the embryos should be at the interface. First instar larvae will sink to the bottom. Rotate slowly at close to horizontal for 20 minutes.
6. After fixation, remove and discard the bottom phase, including larvae that sank to the bottom. Then remove most of the top phase of heptane while leaving the fixed embryos.
7. To remove the vitellin membrane, add a few mLs of fresh heptane. Then fill the test tube 3/4 full of methanol. Cover with a rubber stopper. Vortex for 30 seconds. There should be two phases, a small heptane phase on top and methanol on the bottom.  If there is only one phase, add more heptane and vortex again. Allow embryos to settle. Embryos that sink to the bottom have been devitellinized. Remove everything else. Wash in methanol 3 times. Store in methanol at -20^oC in a tightly sealed container. The freezer should be explosion-proof due to the risk of storing volatile liquids.

 

In Situ Hybridization

Day 1: Embryo Prehybridization Hybridization

All of the prehybridization and hybridization steps are performed in eppendorf centrifuge tubes. Unless otherwise noted, steps were performed at room temperature, although some procedures suggest doing them at 4^oC in order reduce any RNase activity. Embryos should be rotated during washes.

1. Wash embryos 2x in ethanol. Rotate for 1 hr in 1:1 ethanol:xylene at 4^oC.
2. Wash 2x in ethanol. Replace with methanol. Rehydrate with 25%, 50%, 75%, 100% ddH₂O in methanol at 5 minute intervals. (If solution gets cloudy, rehydrate slower)
3. Add 4 volumes of acetone (80% acetone final) and place at -20^oC for 10 minutes.
4. Wash 2x5 min in PBT. Fix for 20 minutes by rotating in PBT containing 4% formaldehyde, followed by 5x5 min washes in PBT.
5. Wash 2x5 min in HB-B. Prehybridize by rotating embryos in 200uL HB-A at 45^oC for 1 hour
6. Add 2uL DIG RNA probe to 100uL fresh HB-A and heat to 80^oC for 5 min. Replace prehyb HB-A with HB-A containing probe and hybridize at 45^oC overnight.

 

Day 2: Washing / Detection

1. Wash 4x30 min in HB-B at 60^oC while rotating.
2. Add 1 volume PBT, followed by 5x5 min PBT washes. Replace with maleic acid buffer (MAB).
3. Rotate embryos for 1 hour in MAB containing 1:2000 Sheep anti-DIG-AP.
4. Wash minimum 5x5 min in MAB. 2 hours of washes recommended.
5. Replace with AMP Staining Buffer 2x5 min. Stain in AMP buffer containing 4.5uL NBT stock and 3.5 uL X-Phosphate stock per mL AMP buffer. Depending on the expression level of the target RNA, the staining may take anywhere from 1 to 6 hours. Wash and store in PBT at 4^oC or non-aqueous storage buffer.

 

Troubleshooting

Probe does not appear on gel / severely degraded
-Shorten probe synthesis reaction from 90 minutes to 60 minutes
-Use fresh polymerase.

High background
-Reduce the concentration of probe used for hybridization
-More washes after antibody

Staining reaction takes too long / Undesirable color
-AMP buffer should be pH 9.2. Normally the reaction stains purplish. Higher pH leads to brownish staining. The pH may also affect the rate of the reaction

   

Low signal
-Use more probe for hybridization
-Replace MAB buffer with PBT in procedure. PBT is a less stringent buffer than MAB.
-Shorten wash times to reduce RNA degradation.