TotalRNAisolationprotocol.docxVIP

Total RNA isolation protocolThe procedure is suitable for all types of tissues from wide variety of animal and plant species. All steps are performed at weak acid pH (HEPES, 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 isolationGuTS extraction buffer:?2 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 (1 mM EDTA, 10 mM Tris-HCl, pH 7.0) or 1xTHE (1 mM EDTA, 2 mM Tris-OH, 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. 2 ml eppendorf tube with tissue sample and glass boll freeze at -80°C, grind in the MM300 Mixer Mill for 5 min at 30 Hz. In 2 ml tube with mechanically disrupted tissue sample add fresh 1ml GuTS extraction buffer, vortex very well and add 500 μl of phenol, vortex very well (shake tube in the MM300 Mixer Mill for 3 min at 30 Hz). Optional: an additional isolation step may be required for samples with high content of polysaccharides or extracellular material, remove insoluble material from the extract by centrifugation at maximum speed on table microcentrifuge for 5 minutes. Transfer the cleared solution to a fresh tube. Incubate the samples at 60°C for 10-30 minutes. Add 300 μl of chloroform-isoamyl alcohol, vortex very well for 2 minute creating an emulsion (in the MM300 Mixer Mill at 30 Hz). Spin at maximum speed on t