Team:UNICAMP-Brazil/Teste

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Isolation of RNA using Trizol Reagent (Invitrogen)
1. Homogenization a. Tissues Homogenize tissue samples in 1 ml of TRIzol® Reagent per 50-100 mg of tissue using a glass-Teflon® or power homogenizer (Polytron, or Tekmar's TISSUMIZER® or equivalent). The sample volume should not exceed 10% of the volume of TRIzol Reagent used for homogenization. b. Cells Grown in Suspension Pellet cells by centrifugation. Lyse cells in TRIzol® Reagent by repetitive pipetting. Use 1 ml of the reagent per 5-10 × 106 of animal, plant or yeast cells, or per 1 × 107 bacterial cells. Washing cells before addition of TRIzol® Reagent should be avoided as this increases the possibility of mRNA degradation. Disruption of some yeast and bacterial cells may require the use of a homogenizer. OPTIONAL: An additional isolation step may be required for samples with high content of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and tuberous parts of plants. Following homogenization, remove insoluble material from the homogenate by centrifugation at 12,000 × g for 10 minutes at 2 to 8°C. The resulting pellet contains extracellular membranes, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA. In samples from fat tissue, an excess of fat collects as a top layer which should be removed. In each case, transfer the cleared homogenate solution to a fresh tube and proceed with chloroform addition and phase separation as described. 2. Phase Separation Incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRIzol® Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 × g for 15 minutes at 2 to 8°C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIzol® Reagent used for homogenization. 3. RNA Precipitation Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIzol® Reagent used for the initial homogenization. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at no more than 12,000 × g for 10 minutes at 2 to 8°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube. 4. RNA Wash Remove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIzol® Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 7,500 × g for 5 minutes at 2 to 8°C. 5. Redissolving the RNA At the end of the procedure, briefly dry the RNA pellet (air-dry or vacuum-dry for 5-10 minutes). Do not dry the RNA by centrifugation under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/280 ratio < 1.6. Dissolve RNA in RNase-free water or 0.5% SDS solution by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60°C. (Avoid SDS when RNA will be used in subsequent enzymatic reactions.) RNA can also be redissolved in 100% formamide (deionized) and stored at -70°C (5). RNA Isolation Notes: 1. Isolation of RNA from small quantities of tissue (1 to 10 mg) or Cell (102 to 104) Samples: Add 800 µl of TRIzol® to the tissue or cells. Following sample lysis, add chloroform and proceed with the phase separation as described in step 2. Prior to precipitating the RNA with isopropyl alcohol, add 5-10 µg RNase-free glycogen (Cat. No 10814) as carrier to the aqueous phase. To reduce viscosity, shear the genomic DNA with 2 passes through a 26 gauge needle prior to chloroform addition. The glycogen remains in the aqueous phase and is co-precipitated with the RNA. It does not inhibit first-strand synthesis at concentrations up to 4 mg/ml and does not inhibit PCR. 2. After homogenization and before addition of chloroform, samples can be stored at -60 to -70°C for at least one month. The RNA precipitate (step 4, RNA WASH) can be stored in 75% ethanol at 2 to 8°C for at least one week, or at least one year at –5 to -20°C.

3. Table-top centrifuges that can attain a maximum of 2,600 × g are suitable for use in these protocols if the centrifugation time is increased to 30-60 minutes in steps 2 and 3.

Troubleshooting

Expected yields of RNA per mg of tissue or 1 x 106 cultured cells

* Liver and spleen, 6-10 µg * Kidney, 3-4 µg * Skeletal muscles and brain, 1-1.5 µg * Placenta, 1-4 µg * Epithelial cells (1 x 106 cultured cells), 8-15 µg * Fibroblasts, (1 x106 cultured cells) 5-7 µg

Low yield

* Incomplete homogenization or lysis of samples * Final RNA pellet incompletely redissolved

A260/A280 ratio < 1.65 RNA sample was diluted in water instead of TE prior to spectrophotometric analysis. Low ionic strength and low pH solutions increase absorbance at 280 nm (6,7).

* Sample homogenized in too small a reagent volume. * Following homogenization, samples were not stored at room temperature for 5 minutes. * The aqueous phase was contaminated with the phenol phase. * Incomplete dissolution of the final RNA pellet.

RNA degradation

* Tissues were not immediately processed or frozen after removal from the animal. * Samples used for isolation, or the isolated RNA preparations were stored at -5 to-20°C, instead of -60 to -70°C. * Cells were dispersed by trypsin digestion. * Aqueous solutions or tubes were not RNase-free. * Formaldehyde used for agarose-gel electrophoresis had a pH below 3.5.

DNA contamination

* Sample homogenized in too small a reagent volume. * Samples used for the isolation contained organic solvents (e.g., ethanol, DMSO), strong buffers, or alkaline solution.

Proteoglycan and polysaccharide contamination

The following modification of the RNA precipitation (step 3) removes these contaminating compounds from the isolated RNA. Add to the aqueous phase 0.25 ml of isopropanol followed by 0.25 ml of a high salt precipitation solution (0.8 M sodium citrate and 1.2 M NaCl) per 1 ml of TRIzol® Reagent used for the homogenization. Mix the resulting solution, centrifuge and proceed with the isolation as described in the protocol. The modified precipitation effectively precipitates RNA while maintaining polysaccharides and proteoglycans in a soluble form. A combination of the modified precipitation with an additional centrifugation of the initial homogenate (note 2, RNA isolation protocol) is required to isolate pure RNA from plant material containing a very high level of polysaccharides.