Team:SDU-Denmark/labwork/Protocols

(The procedure we used for mRNA isolation, is a modified version of the Qiagen RNeasy protocol for plant mRNA isolation.)

Small tubers from Helianthus tuberosus were washed with untreated water and cut in rough slices with an everyday boxcutter. 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. RLT buffer is mixed at this point, for later use. For each mL RLT, add uL beta-mercaptoethanol. Do this in a fume cabinet, as mercaptoethanol smells really bad and is toxic!. 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. The plant powder is transferred to a tube and dissolved in RLT buffer(450uL buffer op 100mg plant material). We used approx. 3mL for 600mg material. The solution is mixed using a vortex mixer. 500 uL of the solution is transfered to a 2mL treated with ultrasound to homogenize the content and further disrupt the cell wall. 500 uL is transfered to a 2mL tube as a control. The samples are centrifuged at 14.500 rpm for 60 seconds to pellet out large organelles. 0,5 vol. 96-100% ethanol is added(250uL) to the sample, mixed by pipette. Transfer the samples to spin-column(Qiagen RNeasy mini kit, for RNA isolation from animal cells) in a 2mL sampletube and do the following:

-spin at 10.000rmp for 15s, discard flowthrough add 700uL RW1 til spin column -spin at 10.000rmp for 15s, discard flowthrough add 500uL RPE buffer til spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol) -spin at 10.000rmp for 15s, discard flowthrough add another 500uL RPE buffer to the spin column (RPE buffer = 1:4 vol RPE/96-100% ethanol) -spin at 10.000rmp for 15s, discard flowthrough Transfer the columns to new 2mL centrifuge tubes. Centrifuge at full speed for 1 min. Transfer spin columns to new 1.5mL sample tubes -add 30-50uL RNase-free water -spin at 10.000rpm for 60 seconds Repeat the prior step once.

The mRNA is now isolated in the 60-100uL at the bottom of the collection tubes

The solutions were tested on Nanodrop:

Control: 627 ng/uL
Sonic: 747 ng/uL

As expected, disruption of the cell wall by ultrasound resulted in a higher yield.