PrimerDigital

The procedure is suitable for all types of tissues from wide variety of animal (and blood) and plant species. All steps are performed at room temperature (RT) (without ice) and without DEPC-treated water. RNA precipitate with lithium chloride (LiCl) for increased stability of the RNA preparation and improvement of cDNA synthesis. The following protocol is designed for small and large tissue samples (tissue volume 10-200 μl), which normally yield about 10-500 μg of total RNA.

Materials for total RNA isolation

• TRIzol Reagent (40% w/w Phenol (saturated at pH 4.3), 1 M guanidine thiocyanate, 1 M ammonium thiocyanate, 0.1 M sodium acetate buffer (pH 5.0), 5% w/w glycerol);
• Chloroform-isoamyl alcohol mix (24:1);
• 100% isopropanol (isopropyl alcohol, 2-propanol);
• 80% ethanol;
• 10 M LiCl;
• Fresh Milli-Q water (or Milli-Q ultrapure BioPak water) or autoclaved 1xTE (0.1 mM EDTA, 10 mM Tris-HCl, pH 7.0). When an ultrafiltration cartridge (BioPak) is utilized at the point-of-use, the water is suitable for genomics applications (quality at least equivalent to DEPC-treated water) and cell culture.

1. 2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball freeze at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz.
2. In 2 ml tube with mechanically disrupted tissue sample add fresh 1 ml TRIzol Reagent, vortex very well, and incubate the sample for 5 minutes at room temperature.
3. Add 0.2 ml of chloroform per 1 ml of TRIzol Reagent used for homogenization. Vortex very well, and incubate the sample for 3 minutes at room temperature.
4. Centrifugate the samples at maximum speed on table microcentrifuge for 5 minutes at +4°C.
5. Transfer the aqueous phase to a fresh microcentrifuge 2 ml tube with an equal volume of chloroform, vortex well. Spin at maximum speed on table microcentrifuge for 5 minutes.
6. Transfer the aqueous phase to a fresh microcentrifuge 2 ml tube with an equal volume of 2-propanol, vortex well. Spin at maximum speed on table microcentrifuge at room temperature for 10 minutes at +4°C. Wash the pellet once with 1.5 ml 80% ethanol. Spin at maximum speed on table microcentrifuge for 5 minutes.
7. Dissolve the pellet (do not dry) in 400 μl 1xTE at 55°С about 10 min, with vortex. Add an equal volume of 10 M LiCl and chill the solution at -20°C for several hours (overnight). Spin at maximum speed on table microcentrifuge for 10 minutes at +4°C. Carefully remove and discard (or save, Fig.1) supernatant (contains: small RNA < 200 nt and DNA). Wash pellet with 1.5 ml 80% ethanol, vortex well, microcentrifuge, discard the ethanol, don't dry the pellet. Dissolve the pellet in 200-400 μl fresh milliQ water (BioPak) or 1xTE.

Load 5 μl of the solution onto a standard (non-denaturing) 1.5 % agarose gel with 1xTHE buffer to check the amount and integrity of the RNA. Add ethidium bromide (EtBr) to the gel to avoid the additional (potentially RNAse-prone) step of gel staining. Load a known amount of DNA in a neighboring lane to use as standard for determining the RNA concentration. Intact RNA should exhibit sharp band(s) of ribosomal RNA.

Notes

1. There is widespread belief that RNA is very unstable and therefore all the reagents and materials for its handling should be specially treated to remove possible RNAse activity. We have found that purified RNA is rather stable and, ironically, too much anti-RNAse treatment can become a source of problems. This especially applies to DEPC-treating of aqueous solutions, which often leads to RNA preparations that are very stable but completely unsuitable for cDNA synthesis. We have found that simple precautions such as wearing gloves (only for your protection from chemicals), avoiding speech over open tubes, using aerosol-barrier tips, and using fresh 1xTE (or 1xTHE) solution (or Milli-Q ultrapure BioPak water) for all solutions are sufficient to obtain stable RNA preparations.
   When an ultrafiltration cartridge (BioPak) is utilized at the point-of-use, the water is suitable for genomics applications (quality at least equivalent to DEPC-treated water) and cell culture. The BioPak cartridges has been validated in Millipore laboratories to warrant the production of pyrogen-free (less than 0.001 Eu/ml), RNAse-free (less than 0.01 ng/ml) and DNase-free (less than 4 pg/μl) ultrapure water, while maintaining both the resistivity and total organic carbon (TOC) of the treated water, it replaces the lengthy diethylpyrocarbonate (DEPC) treatment process to remove nucleases from purified water.
   All organic liquids (phenol, chloroform and ethanol) can be considered essentially RNAse free by definition, as is the dispersion buffer containing guanidine thiocyanate.
2. The volume of tissue should not exceed 1/5 of the extractiom buffer 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 minutes 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 extraction buffer 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.
3. RNA degradation can be assessed using non-denaturing electrophoresis. The first sign of RNA degradation on the non-denaturing gel is a slight smear starting from the rRNA bands and extending to the area of shorter fragments. RNA showing this extent of degradation is still good for further procedures. However, if the downward smearing is so pronounced that the rRNA bands do not have a discernible lower edge, the RNA preparation should be discarded. The amount of RNA can be roughly estimated from the intensity of the rRNA staining by ethidium bromide in the gel, assuming that the dye incorporation efficiency is the same as for DNA (the ribosomal RNA may be considered a double-stranded molecule due to its extensive secondary structure). The rule for vertebrate rRNA - that in intact total RNA the upper (28S) rRNA band should be twice as intense as the lower (18S) band - does not apply to invertebrates. The overwhelming majority have 28S rRNA with a so-called "hidden break". It is actually a true break right in the middle of the 28S rRNA molecule, which is called hidden because under non-denaturing conditions the rRNA molecule is held in one piece by the hydrogen bonding between its secondary structure elements. The two halves, should they separate, are each equivalent in electrophoretic mobility to 18S rRNA. In some organisms the interaction between the halves is rather weak, so the total RNA preparation exhibits a single 18S-like rRNA band even on non-denaturing gel. In others the 28S rRNA is more robust, so it is still visible as a second band, but it rarely has twice the intensity of the lower one.

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Figure 1. LiCl plant RNA precipitation.
1-3: plant DNAs with small RNA (and tRNA, 5S rRNA) from supernatant;
4-6: RNA from pellet LiCl RNA precipitation. Ladder 100 bp.