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| - | (The procedure we used for mRNA isolation, is a modified version of the Qiagen RNeasy protocol for plant mRNA isolation.)
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| - | <p>
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| - | Small tubers from Helianthus tuberosus were washed with untreated water and cut in rough slices with an everyday boxcutter.</br>
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| - | The slices were distributed in cryotubes and weighed, the weights were noted down, and submerged in liquid nitrogen (flash-freeze at -196 °C). Flash-freezing is necessary to halt all RNase activity. </br>
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| - | RLT buffer is mixed at this point, for later use. For each mL RLT, add μL beta-mercaptoethanol. Do this in a fume cabinet, as mercaptoethanol smells really bad and is toxic!</br>
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| - | After 20 minutes 600mg is extracted from the cryotubes, the rest is stored for later use. The 600mg plant material is grinded to a fine dust using a mortar. It is important to keep the plant material frozen during the grinding, to keep the cell wall rigid enough to destroy it.</br>
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| - | The plant powder is transferred to a tube and dissolved in RLT buffer(450μL buffer op 100mg plant material). We used approx. 3mL for 600mg material.</br>
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| - | The solution is mixed using a vortex mixer.</br>
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| - | 500 μL of the solution is transfered to a 2mL treated with ultrasound to homogenize the content and further disrupt the cell wall. 500 μL is transfered to a 2mL tube as a control.</br>
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| - | The samples are centrifuged at 14.500 rpm for 60 seconds to pellet out large organelles.
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| - | 0,5 vol. 96-100% ethanol is added(250μL) to the sample, mixed by pipette. </br>
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| - | Transfer the samples to spin-column (Qiagen RNeasy mini kit, for RNA isolation from animal cells) in a 2mL sampletube and do the following:
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| - | <p>
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| - | -spin at 10.000 rmp for 15s, discard flowthrough
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| - | add 700μL RW1 til spin column <p>
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| - | -spin at 10.000 rmp for 15s, discard flowthrough
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| - | add 500μL RPE buffer til spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol)<p>
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| - | -spin at 10.000 rmp for 15s, discard flowthrough
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| - | add another 500μL RPE buffer to the spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol)<p>
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| - | -spin at 10.000 rmp for 15s, discard flowthrough
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| - | Transfer the columns to new 2mL centrifuge tubes. Centrifuge at full speed for 1 min.
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| - | Transfer spin columns to new 1.5mL sample tubes <p>
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| - | -add 30-50μL RNase-free water <p>
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| - | -spin at 10.000 rpm for 60 seconds <p>
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| - | Repeat the prior step once.
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| - | <p>
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| - | The mRNA is now isolated in the 60-100μL at the bottom of the collection tubes
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| - | <p>
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| | + | </p> |
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