Co-extraction of RNA and DNA from plant tissue

Dominik Buchner

Published: 2023-02-11 DOI: 10.17504/protocols.io.n2bvj8qxngk5/v1

Abstract

This protocol describes how to co-extract RNA and DNA from plant tissue samples. Samples are homogenized and simultaneously lyzed by bead-beating. Cell debris is then caught with a pre-filter column, the DNA is then subsequently bound to a silica column, while the RNA passes the membrane. The RNA in the flow-through is then precipitated with 100% ethanol and bound to a second silica column. Both, DNA and RNA are washed with different wash buffers to remove remaining proteins and other contaminants and finally eluted in separate tubes. If the user is just interested in the RNA, the DNA spin-column can just be discarded.

Before start

Make sure all buffers are prepared before starting.

Steps

Sample preparation and lysis

For each sample prepare one 2 mL screwcap tube pre-filled with approximately 400mg of 2 mm zirconia beads and 0.1 mm glass beads.

Add up to 200mg of plant tissue to the prepared tube.

Note

For samples with a high RNA content less starting material might lead to better results. For most sample types 50mg of starting material will yield a sufficient amount of DNA and RNA for downstream analysis.

Add 800µL to the sample tube.

Note

For complete inactivation and destruction RNAses of 2-Mercaptoethanol can be added in addition. We usually don't because then the samples have to be handled under a fume hood until all lysate has been handled and discarded appropriately.

Immediately bead beat for 0h 5m 0s at maximum speed.

Note

Depending on the bead beater used in this step the time might have to be adjusted. We'd recommend to bead beat the sample until the material is completely homogenized.

Lysate clearing and pre-filtering

Transfer 700µL of the crude lysate to a pre-filter column.

DNA binding

Transfer 700µL of the flowthrough from step 7 to a silica spin column to bind the DNA in the lysate. Keep the flow-through. Mark the spin column as the DNA column.

Note

The protocol will work with all kinds of silica spin columns. See materials section for what we use.

RNA precipitation and binding

Add 350µL to the flow-through from step 8 to adjust the binding conditions to bind RNA to the silica column.

10.

Vortex the samples to mix the lysate with the ethanol. Do not centrifuge.

11.

Load the mixture on a second spin column. Mark this column as the RNA spin column.

11000x g and discard the flow-through.

Note

Two loading steps will be necessary to pass the complete volume through the spin column.

Washing steps

12.

Add 700µL to the RNA spin column ,11000x g and discard the flow-through.

13.

Add 500µL to the RNA spin column , add 500µL to the DNA spin column , 11000rpm and discard the flow-through.

14.

Add 500µL to the RNA spin column , add 500µL to the DNA spin column , 11000rpm and discard the flow-through.

Column drying and elution

15.

11.000rpm to dry the silica membrane of the spin columns. Transfer the spin column to a fresh 1.5 mL microcentrifuge tube.

16.

Add 100µL directly to the silica membrane. Incubate the column for 0h 3m 0s at Room temperature

17.

11.000rpm , store the eluted RNA at -80°C and the eluted DNA at -20°C

Citation

Expected result of the described protocol. Extraction was carried out in 4 replicates, left part of the gel picture shows the DNA fraction of the sample, while the right part shows the RNA fraction.

Co-extraction of RNA and DNA from plant tissue

Abstract

Before start

Steps

Sample preparation and lysis

Lysate clearing and pre-filtering

DNA binding

RNA precipitation and binding

Washing steps

Column drying and elution

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