This protocol is designed for embryos, but can also be used for larvae/adults. Best results have been obtained when looking at RNAs expressed in the adult germline, but some somatic RNAs have also given nice patterns. Modifications necessary to work with larvae/adults are described in section D.
B. Probe synthesis
We have used two types of single-stranded DNA probes: probes derived from cloned cDNAs, and synthetic oligonucleotides. The cDNA-derived probes are used to detect mRNAs derived from specific genes. These probes are synthesised by multiple cycles of primer extension in the presence of digoxigenin dUTP, using a cloned cDNA as a template (eg. "asymmetric PCR", Patel and Goodman, 1992). The oligonucleotide probes are suitable for detecting abundant RNAs containing a defined sequence such as poly-A RNAs (using an oligo-dT probe) and SL1-bearing RNAs (using an anti-SL1 probe). These probes are end-labeled with terminal transferase and digoxigenin-ddUTP.
1. Protocol for preparation of single-stranded probes from cloned cDNA (from Patel and Goodman, 1992):
a) 2-5 µg of plasmid DNA containing the cDNA insert is linearized using an appropriate restriction enzyme. For antisense probes, a unique restriction site 5' to the insert is used. This digested DNA will be amplified using an antisense primer at the 3' end of the insert. For sense (control) probes, a unique restriction site 3' to the insert is used. This digested DNA will be amplified using a sense primer at the 5' end of the insert. (Inserts of up to 2kb are labeled efficiently).
b) Digested DNA is extracted once with phenol/chloroform, once with chloroform, precipitated with 3 volumes of 100% EtOH, and resuspended in TE at a final concentration of 100-200 µg/ml.
c) The following reagents are mixed in 0.5 ml eppendorf tube.
water 7.0 µl
10x Taq Buffer 2.5 µl
25 mM MgCl2 1.5 µl
10 x dNTP mix 5.0 µl
Primer * (30 ng/µl) 5.0 µl
Digested DNA (100-200 µg/ml) 2.0 µl
mineral oil 40 µl
* For cDNAs cloned in Bluscript, we use the following primers (21mers):
"T3" = 5'- ACT AAA GGG AAC AAA AGC TGG -3'
"T7" = 5'- ACT CAC TAT AGG GCG AAT TGG -3'
d. Mixed reagents are boiled for 5 min, before adding 2.0 µl of a 1:8 dilution in water of 5 units/µl Taq polymerase stock (1.25 units total).
e. The labelling reaction is incubated for 35 thermal cycles as follows:
95°C for 45 seconds
55°C for 30 seconds (lower temperature for primers less than 20nt)
72°C for 1 minute
f. 75 µl of H20 is added to the reaction below the oil and 90 - 95 µl of the diluted reaction is transferred to a new tube.
g. 10 µl of 1M NaCl, 10 µg of glycogen, and 3 vols of 100% EtOH are added to the diluted reaction. After 30 min at -70°C, the reaction is centrifuged at 15,000 rpm for 10 min. The pellet is washed in 70% ethanol, dried, and resuspended in 300 µl of hybridization buffer.
h. The probe is boiled for 1-2 hours. This step reduces the length of the probe for efficient penetration of embryos.
e. Probe production is assayed using the following protocol. 1 µl of probe in hybridization buffer is mixed with 5 µl of 5 x SSC, boiled for 5 min, and cooled on ice. 1 µl of this mixture is spotted on a nitrocellulose strip. Several dilutions of a pre-labelled control DNA (1ng to 1pg / µl; Boeringher Mannheim) are also spotted for comparison. The strip is baked for 30 min in a vacuum oven at 80oC, washed once in 2 x SSC, twice in PBT, and blocked for 30 minutes in PBT. The strip is then incubated for 30-60 min with AP-anti DIG antibody diluted 1:2000 in PBT. After three 10 min washes in PBT, and two 5 min washes in staining solution, the strip is developed in staining solution containing 4.5 µl NBT/ml, 3.5 µl X-phosphate/ml. Spots should be visible within minutes. Spot intensities of the probe and control dilutions are compared to determine the concentration of the probe.
f. Probes can be stored at -20oC in hybridization buffer for several weeks.
2. Synthesis of oligonuceotide probes:
Synthetic oligonucleotides are end-labelled using terminal transferase and digoxigenin-ddUTP (Boeringher Mannheim sells an 3' end-labeling kit [Cat # 1362372]; we have used these reagents on gel-purified oligonucleotides). Labeled oligonucleotide probes are resuspended to 0.5 µg/ml in hybridization buffer. The percentage of formamide and concentration of SSC in the hybridization buffer are adjusted for each oligonucleotide to give a melting temperature (Tm) of 52oC, using the following formula (Davis et al., 1986):
Tm = 16.6 log[M] 0.41[Pgc] 81.5 - B/L - 0.65[Pf]
where M is the molar concentration of Na (maximum of 0.5), Pgc is the percent of G and C bases in the oligonucleotide, B is 675 for synthetic oligos up to 100 bases in length, L is the length of the probe in bases, and Pf is the percent concentration of formamide. A Tm of 52oC allows for efficient hybridization at 37oC.
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