Team:UNICAMP-Brazil/Protocols/RNA isolation from 'Gallus gallus' spleen, blood and oviduct
From 2009.igem.org
(New page: 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 p...) |
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- | 1.Homogenization | + | [[Team:UNICAMP-Brazil/Protocols|Back to Protocols]] |
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+ | ==Isolation of RNA using Trizol Reagent (Invitrogen)== | ||
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+ | 1. Homogenization | ||
a. Tissues | 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. | 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 | + | 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 × | 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. | 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. | ||
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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. | 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 | + | |
+ | '''Troubleshooting''' | ||
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Expected yields of RNA per mg of tissue or 1 x 106 cultured cells | Expected yields of RNA per mg of tissue or 1 x 106 cultured cells | ||
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- | + | * 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 | ||
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Low yield | Low yield | ||
- | + | * Incomplete homogenization or lysis of samples | |
- | + | * Final RNA pellet incompletely redissolved | |
- | A260/A280 ratio <1.65 | + | 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). | 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 | 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 | 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 | 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. | 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. | ||
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+ | {{:Team:UNICAMP-Brazil/inc_rodape}} |
Latest revision as of 18:01, 24 September 2009
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