Preparation of RNA

For the preparation of embryo tissue RNA, we routinely use a commercial reagent called RNA STAT-60. This procedure is based on the method described by Chomczynski and Sacchi (11), but the RNA STAT-60 reagent contains the guanidine thiocyanate together with the phenol for easier handling. Other manufacturers supply similar reagents yielding identical results (see Note 10).

1. Embryonic tissue should be collected and washed in ice-cold PBS before addition of the extraction reagent. If the tissue of interest has to be collected over a long period of time, the tissue should be rinsed in PBS and flash-frozen into a tube inserted in a tank containing liquid nitrogen. Transferring of PBS to the freezing tube is not desirable. Samples can be stored frozen at -80°C.

2. Once a desired amount of tissue has been collected, the extraction solution should be added directly to the frozen tissue. Typically 4 vol of RNA STAT reagent are added/vol of sample in a 15-mL conical polypropylene graduated tube. Homog-enization of the tissue with the chaotropic solution should be done as fast as possible. For this purpose, a Polytron is essential. The isolation of intact RNA greatly depends on the speed of this step. For small amounts of tissue, the homogeneization can be performed in a microcentrifuge tube: add the solution and shear the sample passing all the material through a 21-gage needle three or four times, until the material easily flows through the needle.

3. After homogeneization, 0.2 mL of chloroform are added/mL of RNA STAT solution used. The tubes are left at room temperature for 5 min.

4. Spin down the tubes at maximum speed in a tabletop centrifuge for 10 min. Alternatively, the mix can be transferred to 1.5-mL microfuge tubes that can be spun in a microcentrifuge.

5. Transfer the aqueous phase to 1.5-mL tubes (a maximum of 700 pL/tube), and add 700 pL of isopropanol. Precipitate for 1 h at room temperature. These tubes can also be stored at -20°C until needed.

6. Spin for 10 min, discard the supernatant, and rinse the RNA pellet with 70% ethanol to eliminate traces of salts and phenol. Once the ethanol has been completely removed (spin the tube shortly and remove the remaining liquid with a yellow tip), the pellet is resuspended in a minimal volume of nuclease-free water. Do not let the pellet dry, or resuspension will be very difficult.

7. Once resuspended, take a small volume of the sample, dilute it in a volume of water according to the size of the spectrophotometer quartz cuvet available, and measure the absorbance at 260 nm. Consider that 1 OD = 40 mg/mL of RNA. Absorbance at 280 nm should indicate contamination with proteins and/or phenol. A good-quality RNA should have a ratio of OD26o/OD28o =1.8-2.0.

8. Load an amount equivalent to 1 pg of total RNA in a regular freshly made 0.8% agarose-1X TBE gel containing 0.2 pg/mL ethidium bromide to check the integrity of the RNA. Two major bands corresponding to the 28S and 18S ribosomal RNAs should appear clear and sharp.

9. Once the RNA has been checked, DNase treatment is required to remove DNA that will interfere with the PCR process, giving rise to bands that do not correspond to cDNA. Resuspend part of the RNA solution (5-50 pg) in 42 pL of nu-clease-free water and add 5 pL of any clean 10X restriction enzyme buffer, 2 pL of DNase I RNase-free, and 2 pL of RNase guard. Incubate at 37°C for 30 min. Store the rest of the RNA at -80°C.

10. Add 50 mL of 2X PK buffer and 5 mL of 10 mg/mL PK. Incubate at 37°C for 30 min.

11. Extract twice with 100 mL of Phenol-SEVAG, recover the aqueous phase, and add 250 mL of 100% ethanol. Precipitate at least 30 min at room temperature. Do not precipitate at a cold temperature, since an excess of SDS and salt can also precipitate and interfere with subsequent steps.

12. Spin down the precipitate, rinse with 70% ethanol, remove all the remaining liquid after a 5-s spin, and resuspend the pellet in a small volume of nuclease-free water. Measure the absorbance at 260 nm, and resuspend the RNA at 100 ng/mL. Integrity of the RNA should be checked at this point as mentioned above. To minimize repeated freezing and thawing, the RNA should be stored in small aliquots at -80°C. The integrity of the RNA is crucial for the success of the whole procedure.

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