Notes

1. Inject 0.02 nmol (27 nL of a 6-ng/nL solution) of c-mos antisense per oocyte. Then, inject the RNAs of interest in both c-mos-injected and noninjected oocytes and treat with progesterone (10 | M) as required. Oocytes injected with c-mos antisense fail to undergo germinal vesicle breakdown, and a white spot is not observed, whereas control oocytes matured normally in response to progesterone.

2. It is preferable to linearize with restriction enzymes that produce a 5' overhang because enzymes leaving 3' overhanging ends may result in low levels of transcription (37).

3. Confirm that cleavage is complete by resolving an aliquot of the restriction reaction in a 1% agarose gel electrophoresis. Even a small amount of circular plasmid in a template preparation will generate a large proportion of incorrect transcripts.

4. An incubation for 5 to 10 min at 65 to 70°C may be required to resuspend the RNA.

5. Quantification can also be obtained by adding trace amounts of a radiolabeled NTP to the RNA transcription, followed by trichloroacetic acid (TCA) precipitation, filtration through a Whatman GF/C glass fiber filter, and measurement in a scintillation counter (33).

6. If necessary, the full-length mRNAs can be purified by cutting out the RNA band, crushing the gel slice in 0.5 M ammonium acetate and 1 mM EDTA, and incubating for 3 h at room temperature. The gel is separated from the eluate by a brief spin, and the RNA is recovered as described in Subheading 3.2.2., step 4, with the addition of 1 ||L of glycogen (20 mg/mL) as a carrier.

7. RNAs may be denatured by treatment with 2.5 M formaldehyde and 50% formamide in 1X MOPS (2 mM sodium acetate, 1 mM EDTA, 20 mM MOPS (3-(N-morpholino)-propanesulfonic acid), pH 7.0, and 10 ng/|L ethidium bromide and heated 10 min at 70°C. Samples are run in agarose gels containing 2.2 M formaldehyde and 1X MOPS.

8. The minimum amount of RNA that can be detected with ethidium bromide is on the order of 20 to 50 ng. Avoid the xylene cyanol and bromophenol blue dyes in the gel-loading buffer because they comigrate with the luciferases mRNAs, interfering with the quantification.

9. The presence of dispase allows the use of a lower collagenase concentration. It is advisable to select the oocytes the day before the experiment and leave them overnight in 1X MBS at 20°C. Manual isolation of the oocytes is also possible, and the resulting oocytes have a higher rate of protein synthesis (38).

10. RNAs on the nanomolar order are very susceptible to degradation and cannot be tested for integrity. Therefore, it is recommended to discard them after the injection and prepare new dilutions from the stock for each experiment.

11. For microinjection, the needle should be 50 to 75 mm long, and the tip must have steep shoulders followed by a gradual taper approx 5 to 10 mm long. The gradual taper allows the tip to be broken repeatedly to adjust the orifice or to reopen a clogged tip.

12. Progesterone adheres to plastic, and even traces of progesterone could induce oocyte maturation. Thus, plastic dishes or pipets should not be reused, and glassware must be cleaned thoughtfully.

13. Contamination with the lipid phase does not affect the luciferase activity. However, contamination with pigment granules must be avoided. If necessary, a second clearing step can be performed. Samples can be stored at -80°C for up to 1 mo.

14. Because, on average, 80 to 100 adenines are added to the polyadenylated RNAs, for probes longer than 300 nucleotides it becomes more difficult to detect differences in size caused by poly(A) tail elongation. If longer 3'-UTRs are studied, they can be shortened prior to electrophoresis by incubation with an antisense oligonucleotide followed by RNase H treatment, as described in ref. 29.

15. Before loading the sample, flush out the urea from the wells with a syringe and running buffer (1X TBE). A 100-nucleotide RNA comigrates with xylene cyanol.

Acknowledgments

We are grateful to Fátima Gebauer for the PBSK-A construct, the luciferase cDNAs, and advice with the dual-luciferase assay. We also thank Mercedes Fernandez for her assistance. Our work in the translational control and cytoplasmic polyadenylation of cyclin B1 mRNA is supported by the Ministerio de Ciencia y Tecnologia (SAF2002-03201), by the Fundación La Caixa (0N03-13-0), and by the Fundación La Maratón de TV3.

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