The procedure is suitable for all types of tissues from wide variety of animal (and blood) and plant species. All steps are performed at weak acid pH (MOPS or MES free acids) and 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

• GuTC extraction buffer: 2.5 M guanidine thiocyanate, 0.1 M LiCl, 10 mM EDTA, 0.1 M MOPS, pH 4.6;
• Phenol, pH 4.5-6.6;
• Chloroform-isoamyl alcohol mix (24:1);
• 100% isopropanol (isopropyl alcohol, 2-propanol);
• 70% 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) or 1xTHE (0.1 mM EDTA, 2 mM Tris, 8 mM HEPES, 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 tube with tissue sample and glass boll freeze at -80°C, grind in the MM300 Mixer Mill for 5 min at 30 Hz.
2. In 2 ml tube with mechanically disrupted tissue sample add fresh 1.5 ml GuTC extraction buffer, vortex very well, and incubate the samples at 60°C for 10-30 minutes. Spin at maximum speed on table microcentrifuge for 5-10 minutes.
3. Transfer 1 ml of the supernatant (the pellet contains polysaccharides and high molecular weight DNA) to a fresh tube with 500 μl of phenol, vortex very well and incubate for 5 minutes.
4. Add 400 μl of chloroform-isoamyl alcohol, vortex very well for 1 minute creating an emulsion (or in the MM300 Mixer Mill at 30 Hz). Spin at maximum speed on table microcentrifuge for 3 minutes at RT.
5. Transfer the aqueous phase to a fresh microcentrifuge 2 ml tube with 700 μl of chloroform-isoamyl alcohol and vortex well (in the MM300 Mixer Mill for 2 min at 30 Hz). Spin at maximum speed on table microcentrifuge for 2 minutes at RT.
6. Transfer the aqueous phase to a fresh microcentrifuge 2 ml tube with an equal volume of 2-propanol and mix well. Spin at maximum speed on table microcentrifuge at room temperature for 2 minutes. Wash the pellet once with 1.5 ml 70% ethanol. Spin immediately at maximum speed on table microcentrifuge at room temperature for 2 minutes.
7. Dissolve the pellet (do not dry) in 400 μl 1xTE at 55°С about 10-20 min, with vortex.
8. Optional: add an equal volume of 10 M lithium chloride, mix well, and chill the solution at -20°C for several hours (overnight). Spin at maximum speed on table microcentrifuge for 10 minutes. Carefully remove and discard (or save, Fig.1) supernatant (contains: small RNA < 200 nt and DNA). Wash pellet with 1 ml 70% 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 0.5x TBE 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 .................More Read....