http://www.eeob.iastate.edu/faculty/WendelJ/rnaextraction.htm
Microscale Hot Borate RNA Extraction from Cotton Tissue
Wendel Lab
Department of Ecology, Evolution and Organismal Biology
Iowa State University, Ames, IA 50011, USA
Prior to Extractions
· Bake all necessary glassware, metal spatulas, mortars, and pestles overnight in a 200°C oven after wrapping them in aluminum foil. For each sample, you should minimally prepare one mortar and pestle, one homogenizer and one spatula.
· DEPC-treat a sufficient volume of water for all dilutions (500 mL is usually plenty).
· Prepare all reagents and solutions.
Day 1
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Place all mortars to be used in liquid nitrogen to cool. Heat homogenizers to 65°C and acquire dry ice. After the mortars are cooled, nestle them in the dry ice.
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Collect 1 g of tissue and flash freeze in liquid nitrogen.
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Transfer to a cooled mortar and pestle. Grind tissue in liquid nitrogen until it is a fine powder.
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Remove one pre-warmed glass homogenizer. Add 8 mL of the 85°C XT buffer and 10 µL of Proteinase K solution (10 mg/mL). Transfer the frozen, ground sample to the warm XT buffer and grind thoroughly until the suspension is evenly dispersed.
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Transfer homogenate to a labeled 50 ml Oakridge tube. Mix and incubate for 1.5 hours in the 42°C incubator/shaker.
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Remove from incubation. Add 1040 µL of 2M potassium chloride (for a final concentration of 160 mM; this step will precipitate proteins from the extract). Gently vortex to mix. Incubate on ice for 1 hour.
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Centrifuge at 5000 rpm for 20 minutes at 4°C to remove debris.
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Transfer the supernatants to new, labeled 50ml Oakridge tubes.
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Add 1/3 volume of 8M lithium chloride (making a final concentration of 2M). Incubate on ice overnight.
Day 2
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Place tubes in the pre-chilled centrifuge and chilled rotor. Spin at 10,000 rpm (12,000 g) for 20 minutes at 4°C. Decant and discard supernatant.
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Wash pellet in 1.5 mL of ice-cold 2M lithium chloride. Disperse pellet with a sterile disposable pipette (as needed) after adding the lithium chloride. This will minimize the retention of unwanted substances.
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Centrifuge tubes at 10,000 rpm (12,000 g) for 15 minutes at 4°C. Decant and discard supernatant.
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Repeat steps 2 & 3 twice more. Spin for 6 minutes.
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Suspend pellet in 250 µL of 1X TE, ph 8.0 and gently vortex. The sample may be warmed to room temperature to facilitate diffusion.
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Remove the insoluble material by centrifuging tubes at 10,000 rpm (12,000 g) for 10 minutes at 4°C.
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Save the supernatant and transfer it to pre-labeled 1.5 mL microcentrifuge Eppendorf tubes. Add 1/10 volume (about 28 µl) of 2 M potassium acetate (pH 5.5). Incubate on ice for 15 minutes. This will removed positively-charged polysaccharides, residual proteins, and other salt-insoluble material.
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Centrifuge tubes at 10,000 rpm (12,000 g) for 15 minutes at 4°C. Discard pellet by transferring supernatant to a new tube.
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Precipitate RNA with 1/10 volume (about 31 µL) of 3 M sodium acetate (pH 6.0) and 2.5X volume (about 875 µL) of cold 100% ethanol. Store at -80°C for 1-2 hours.
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Centrifuge at 13,000 rpm for 20 minutes at 4°C to collect RNA. Remove ethanol and discard. Wash with 1 mL of 70% ethanol. Centrifuge for 5 minutes at 4°C. Aspire or pipette off the ethanol.
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