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INFORMATION FOR

In Situ Hybridization - Making Antisense RNA Probes (dig labelled)

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In vitro transcription in the direction opposite to in vivo transcription of the mRNA makes an antisense RNA that is complementary to the mRNA. This antisense probe can them be used for whole mount in situ hybridizations. The RNA probe is synthesized in the presence of a digoxigenin-substituted nucleotide, dig-UTP. Please refer to references for further information.

Linearize Probe: (1 hour - 2 hour incubation)

Set up digests in eppendorf tube
(100 µL total):

___ 40 µl DNA template (~0.5-1mg/ml)
___ 20 µl restriction enzyme buffer
___ 1 µl restriction enzyme
___ distilled H2O to 100 µl
___ + µL BSA*

*if necessary for enzyme

___ React at 37oC for 2 hours at least

The DNA template (plasmid cDNA) is linearized with a restriction enzyme. The DNA should be cut in a way such that RNA polymerase will fall off at the end of the gene of interest (5' end of gene).
To ensure that the plasmid cDNA is cut properly, run a diagnostic gel. Run 1 µL of digested DNA with 15 µL 1x loading buffer. Also load a sample of undigested for comparison. Look for clean cuts (linearized strand of DNA). Probes of at least 300 bp are necessary for good signal.

After linearizing, storage of linearized DNA is possible for later use. Store at -20oC.

Clean DNA on RNase free PCR purification kit: (~30 min)

*It is essential to keep everything RNAse free. RNA is degraded relatively easily. Wear gloves at all times. All reagents should be RNAse free.

___Add 500μl buffer PB to sample, mix

___ Apply sample to column, spin 1 min @ 13000 rpm

___ Discard flow through, add 750 µl buffer PE, spin 1 minute.

___ Discard flow through and spin 1 additional minute to dry column completely.

___ Place column in RNAse free eppendorf tube.

___Apply 20 - 30 µl NFW, incubate on bench 1 min, spin 1 min.

___ Place column on a second eppendorf tube, and apply a second fresh aliquot of NFW to column.

___ Incubate on bench 1 min, spin 1 min.

___ Use 1 µl to Nanodrop, run 1 µl on clean .8% gel.

Don't forget to use RNAse free reagents. If you suspect a low yield, elute in laess NFW...you can always add more. Also you can incubate the water at 60 - 70 degrees before applying to column.

To ensure that the DNA was not lost in the cleaning procedure, run out 1 µL of DNA solution on a gel.

DNA probe template should be stored at -20oC. Template can be stored for long periods of time.

Make RNA Probe: (~1 hour until 2 hr. incubation)

Incubate the following reaction at 37oC for 2 hours. Be sure to keep everything RNAse free. When making multiple probes, it is best to make a mix of reagents, then aliquot the this each tube. Certain reagents, including enzyme and template, should be kept on ice at all times to prevent degradation.
Reagent Amount

Volume

in Reaction

Mix Volume/Notes
1. RNAse free H2O Fill to 20 µL
2. 10x Transcription Buffer 2 µl
3. dig-UTP mix 1 µl
4. Enzyme (Polymerase) 2 µL
DNA 1µg _______µl

Remove DNA template: (~0.5 hour until LiCl precipitation)

___ Add 1 µl of 1 mg/ml RNAse free DNAseI and incubate at 37oC for 10 minutes. The DNAseI will degrade the DNA template into single nucleotides which will stay in solution even during the following LiCl precipitation. Keep the DNAseI on ice.

___ Add 30 µl of LiCl.

Precipitate RNA by incubating at -20oC for at least 1 hour up to overnight.

Alternately, use Zymo Clean up kit and skip the following 5 steps.

The LiCl will precipitate the RNA probe. Most people incubate overnight at -20 for maximum yeild.
Thaw 40mM Sodium Bicarbonate/60 mM Sodium Carbonate if you want to hydrolyze probe.

___ Centrifuge 10 minutes @ 4 degrees to pellet RNA.

Keep your tube cold until re-suspension step.
___ Remove supernatant Be careful not to remove the pellet.
___ Wash with 1 ml RNAse free 70-80% EtOH The ethanol wash will remove the residual LiCl.
___ Remove supernatant Be careful not to remove the pellet.
___ Resuspend in 20 µl dep H20
___ Measure Concentration by Nanodrop

___ Resuspend in Hyb Buffer at a 10X concentration- 10 µg/ml or 100X 100 µg/ml

NOTE: Although the probe will dissolve quickly in dep H20, it dissolves considerably more slowly in Hyb Buffer. Therefore, check to ensure that it is dissolved.

To Make Dig Mix

to 25 µl of 10 mM dig-UTP
add:
10 µl ATP 100 mM
10 µl GTP 100 mM
10 µl CTP 100 mM
7.5 µl UTP 100 mM

Reagents:
T7 High Yield RNA Synthesis Kit
E2040S 50 reactions

Dig-UTP
11209256910
25 µl 250 nmol (10 mM)

Contributed by Mustafa K. Khokha, 06/2011.

References

Sive, HL, Grainger RM, Harland RM; Early Development of Xenopus Laevis: A Laboratory Manual; Cold Spring Harbor Laboratory Press; New York, 2000; pp. 249-297.