Preparation of RNA

The RNA for microinjection is synthesised by in vitro transcription. The structure of the 5'- and 3'-termini of synthetic mRNA can affect its stability and the efficiency of translation. Capping of the 5' terminus with GpppG and addition of the poly(A) tail improve the stability of synthetic mRNAs. The constructs used widely to generate the DNA templates for in vitro transcription are pSP64T and CS2 vectors (6-9). The synthetic RNA should be tested in an in vitro translation system to ascertain that a protein product is made.

1. Prepare 10 | g of template DNA by digestion with a suitable restriction enzyme. Make sure the reaction is completed by analyzing an aliquot of the DNA digest in an agarose gel.

2. Purify the linear DNA by extraction with phenol/chloroform and precipitation with ethanol. Redissolve the DNA at 0.5 mg/mL in RNase-free water (Sigma, W-4502) or DEPC-treated water.

3. Mix the following components at room temperature in the order shown:

4.0 |L DNA template.

19.5 |L RNase-free water.

2.0 |L 0.1 M dithiothreitol.

4.0 |L 10X transcription buffer.

2.0 |L placental RNase inhibitor (10 U/mL).

2.0 |L DNA-dependent RNA polymerase.

4. Incubate for 2 h at 37°C. Analyze an aliquot of the reaction (2 |L) on an agarose gel to check the transcription efficiency.

5. Add 1 |L of RNase-free DNase I (1 mg/mL), mix, and continue the reaction for 15 min at 37°C.

6. Add 160 |L of RNase-free water and purify the RNA by phenol/chloroform extraction twice.

7. Further purify the RNA by the microconcentrator as described above.

8. Check the size and quality of RNA by gel electrophoresis. Aliquot the RNA, and store at -80°C.

9. The amount of RNA for microinjection varies according to the nature of its encoded protein. It is, therefore, necessary to titrate the RNA.

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