Total RNA isolation protocol

We suggest the following procedure for total RNA isolation based on years of experience working with complicated samples and small amounts of starting material (Matz, 2002). The procedure is suitable for all types of tissues from wide variety of animal species. The method is based on the well-known protocol of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987), except that all steps are performed at neutral pH instead of acidic as was originally suggested. Also, we precipitate the RNA with lithium chloride (LiCl) for increased stability of the RNA preparation and improvement of cDNA synthesis.

As an alternative, the popular Trizol method (GIBCO/Life Technologies) may be used, although it does not work on some non-standard species such as jellyfish. Kits for RNA isolation that utilize columns (such as Qiagen''s RNeasy kit) are generally not recommended for non-standard samples.

The following protocol is designed for large tissue samples (tissue volume 10-100 μl), which normally yield about 10-100 μg of total RNA. Smaller amounts of starting material (expected to yield about 1 μg RNA or less) should be prepared in the same way, with the exclusion of the second phenol-chloroform extraction (step 4) and LiCl precipitation (step 6). Additionally, the final "pellet" should be dissolved in 5 μl instead of 40 μl of water and transferred directly to cDNA synthesis, omitting the agarose gel analysis.

Materials for total RNA isolation

Note: Simple precautions such as wearing gloves, use of aerosol-barrier tips, and fresh sterile water for all solutions are sufficient to obtain stable RNA preparations. All organic liquids (phenol, chloroform and ethanol) can be considered essentially RNAse free by definition, as is the dispersion buffer containing 4 M guanidine thiocyanate. We do not recommend that you use DEPC-treated aqueous solutions as such treatment often leads to RNA preparations that are very stable but completely unsuitable for cDNA synthesis.

Commonly, genomic DNA contamination does not affect cDNA synthesis. DNase treatment to degrade genomic DNA is not recommended. In some cases, excess of genomic DNA can be removed by LiCl precipitation or by phenol:chloroform extraction.

- Dispersion buffer ("buffer D"): 4M Guanidine thiocyanate, 30 mM disodium citrate, 30 mM β-mercaptoethanol, pH 7.0-7.5

Note: Normally the dispersion buffer does not require titration. If the pH is significantly lower than 7.0, try another batch of guanindine or disodium citrate. The buffer may be stored for years at +4°C in the dark.

- Buffer-saturated phenol, pH 7.0-8.0 (GIBCO/Life Technologies).

- Chloroform-isoamyl alcohol mix (24:1).

- 96% ethanol.

- 80% ethanol.

- 12 M lithium chloride.

- Co-precipitant (optional): see DNA reagent (Amersham) or glycogen.

- Fresh steril water (e.g. milliQ-purified)

- Agarose gel (1%) containing ethidium bromide.

Total RNA Isolation Protocol

1. Dissolve the tissue sample in buffer D.

Note: The volume of tissue should not exceed 1/5 of the buffer D volume. To avoid RNA degradation, tissue dispersion should be carried out as quickly and completely as possible, ensuring that cells do not die slowly on their own. To adequately disperse a piece of tissue usually takes 2-3 min of triturating using a pipet, taking all or nearly all volume of buffer into the tip each time. The piece being dissolved must go up and down the tip, so it is sometimes helpful to cut the tip to increase the diameter of the opening for larger tissue pieces. Tissue dispersion can be performed at room temperature. The tissue dispersed in buffer D produces a highly viscous solution. The viscosity is usually due to genomic DNA. This normally has no effect on the RNA isolation (except for dictating longer periods of spinning at the phenol-chloroform extraction steps), unless the amount of dissolved tissue was indeed too great. In some cases (for example, freshwater planarians or mushroom anemones), mucus produced by the animal contributes to viscosity. This substance tends to co-purify with RNA, making it very difficult to collect the aqueous phase at the phenol-chloroform extraction step. It likewise lowers the efficiency of cDNA synthesis. The RNA sample contaminated with such mucus, although completely dissolved in water, does not enter agarose gel during electrophoresis. The EtBr-stained material stays in the well, probably because the mucus adsorbs RNA. We have found that including cysteine in buffer D can diminish the mucus problem. To buffer D add 0.1 volume of solution containing 20% cysteine chloride and 50 mM tricine-KOH, pH 7 (takes a lot of titration!). The cysteine solution should be freshly prepared. After dissolving the tissue, incubate the sample for 2 hours at +4°C, and then proceed with the above protocol.

2. Spin the sample at maximum speed on a microcentrifuge for 5 min at room temperature to remove debris. Transfer the supernatant to a new tube.

3. Put the tube on ice, add an equal volume of buffer-saturated phenol and mix. There will be no phase separation at this time. Add 1/5 volume of chloroform-isoamyl alcohol (24:1) and vortex the sample. Two distinct phases will separate. Vortex three to four more times with about 1 minute between steps. Incubate the tube on ice between steps. Spin at maximum speed on table microcentrifuge for 30 min at +4°C. Remove and save the upper, aqueous phase. Avoid warming the tube with your fingers or the interphase may become invisible.

4. Repeat step 3.

5. Add 1 μl of co-precipitant, and then add one volume of 96% ethanol and mix. Spin immediately at maximum speed on table microcentrifuge at room temperature for 10 min. The precipitate may not form a pellet, being instead spread over the back wall of the tube and thus being almost invisible even with co-precipitant added. Wash the pellet once with 0.5 ml 80% ethanol. Dry the pellet briefly until no liquid is seen in the tube (do not over-dry).

6. Dissolve the pellet in 100 μl fresh milliQ water. If the pellet cannot be dissolved completely, remove the debris by spinning the sample at maximum speed on table microcentrifuge for 3 minutes at room temperature. Transfer the supernatant to a new tube, then add an equal volume of 12 M LiCl and chill the solution at -20°C for 30 min. Spin at maximum speed on table microcentrifuge for 15 min at room temperature. Wash the pellet once with 0.5 ml 80% ethanol, and dry as previously done. The precipitated RNA is usually invisible, since co-precipitant does not precipitate in LiCl.

7. Dissolve the pellet in 40 μl fresh sterile water.

8. After RNA isolation, we recommend RNA quality estimation using gel electrophoresis. Denaturing formaldehyde/agarose gel electrophoresis should be performed as described (Sambrook et al., 1989 ). Alternatively, standard agarose/ethidium bromide (EtBr) gel electrophoresis can be used to quickly estimate RNA quality (see recommendations to perform a non-denaturing agarose gel electrophoresis of RNA.

9. To store the isolated RNA, add 0.1 volume of 3 M sodium acetate and 2.5 volumes 96% ethanol to the RNA in water, and mix thoroughly. The sample may be stored for several years at -20°C.

References

• Chomczynski P, Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159.
• Matz M.V. (2002) Amplification of representative cDNA samples from microscopic amounts of invertebrate tissue to search for new genes. Methods Mol Biol. 183: 3-18 / pmid: 12136765.
• Sambrook J., Fritsch E.F. and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.