Mar 08, 2023
QIAGEN RNeasy Plant RNA Extraction Protocol (Modified)
This protocol is a draft, published without a DOI.
- 1University of Illinois at Urbana-Champaign
Protocol Citation: Steven J Burgess 2023. QIAGEN RNeasy Plant RNA Extraction Protocol (Modified). protocols.io https://protocols.io/view/qiagen-rneasy-plant-rna-extraction-protocol-modifi-cqrjvv4n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 08, 2023
Last Modified: August 14, 2023
Protocol Integer ID: 78347
Abstract
This is a protocol for extraction of RNA from plant leaf tissue using a QIAGEN RNeasy Plant Mini Kit. The procedure largely follows the manufacturer's instructions but there are a few minor tweaks we introduced which in our hands were necessary for optimal results. This protocol also includes the on-column DNase I digestion step.
The original protocol is attached below
HB-0572-002 1101268_PCard_RNY_Plant_Mini_0316_WW_WEB.pdf Kit:
RNase free DNase kit:
Guidelines
It is critical that you do not overload the spin columns to ensure high purity of final RNA. Therefore before sampling determine how much an individual leaf disk weighs then harvest and freeze only 100 mg or less of fresh leaf tissue per sample vial.
Materials
- 2 mL centrifuge tubes
- Humboldt brass cork borer set (07-865-10B; Fisher Scientific)
- 13.4 mm diameter, flash-frozen leaf disks
- RNeasy Plant Mini Kit (QIAGEN; 74904)
- RNase-Free DNase Set (QIAGEN; 79254)
Safety warnings
Perform all steps within a fume hood and collect tips and tubes in thehazardous material collection bins. β-mercaptoethanol (β-ME) included in the extraction buffer is toxic, harmful to the environment and corrosive (it also stinks!)
Before start
- Calculate the volume of extraction buffer RLT required for the number of samples to be processed. Each sample requires at least 450 µL working on the assumption that 100 mg of fresh tissue is disrupted. It is advisable to prepare 500 µL of RLT per sample, to ensure sufficient buffer is made. (So for 10 samples, this would be 5 mL )
- To prepare RLT buffer, in a fresh tube add 10 µL β-ME per 1 mL Buffer RLT.
- Add 4 volumes of ethanol (96–100%) to Buffer RPE for a working solution
- Prepare DNase I stock solution before using the RNase-Free DNase Set for the first time. Dissolve the lyophilized DNase I (1500 Kunitz units) in 550 µl of the RNase-free water provided. To avoid loss of DNase I, do not open the vial. Inject RNase-free water into the vial using an RNase-free needle and syringe. Mix gently by inverting the vial. Do not vortex (DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube.)
- For long-term storage of DNase I, remove the stock solution from the glass vial, divide it into single-use aliquots, and store at –20°C for up to 9 months. Thawed aliquots can be stored at 2–8°C for up to 6 weeks. Do not refreeze the aliquots after thawing.
- Calculate the volume of DNase I working solution required for the number of samples to be processed. Each sample requires 80 µL . Make a master mix containing 10 µL of DNAse I per sample and70 µL buffer RDD. It is advisable to make up 10-20% extra than you need to account for pipetting inaccuracies. (So for 10 samples this would be 120 µL of DNAse I and 840 µL of buffer RDD). Prepare in a new nuclease-free tube
Collect samples immediately into liquid nitrogen and store at -80C. Grind per "Grinding Tissue with the Qiagen Tissuelyzer".
Loading samples
Loading samples
Add 450 µL Buffer RLT to a maximum of 100 mg tissue powder. Vortex immediately until the powder is re-suspended.
Note
This step can take up to a couple of minutes. If lumps form it can lead to partial thawing before the powder is completely dissolved in the buffer and degradation of RNA due to the activity of endogenous RNAses. It is therefore advisable to start with samples that are at -70 °C , (tubes on dry ice) rather than moving samples straight from LN2, which has a greater tendency for lumps to form.
It is critical to pre-weigh your samples so as not to overload the spin columns, using too much tissue will result in sub-standard purity of RNA after extraction
Spin samples 12000 x g, Room temperature, 00:01:00 to pellet any residual debris.
Note
This is not included in the original protocol, ideally following proper grinding and re-suspension there should be little to no debris or clumps. However, in our experience, there can be aggregates of insoluble material, spinning will help keep these to the bottom of the centrifuge tube
Transfer the lysate to a QIAshredder spin column (lilac) placed in a 2 mL collection tube
Note
Be careful not to disturb any pellet, if you are having trouble with the tip getting blocked it can help to cut off the end
Spin 12000 x g, Room temperature, 00:02:00
Transfer the supernatant of the flow-through to a new microcentrifuge tube (not supplied) without disturbing the cell-debris pellet.
Note
Use nuclease-free centrifuge tubes
Add 0.5 volume of ethanol (96–100%) to the cleared lysate, and mix immediately by pipetting. Do not centrifuge.
Note
Typically the volume of EtOH to use will be 225 µL
Transfer the sample (usually 650 µL ), with any precipitate, to an RNeasy Mini spin column (pink) in a 2 mL collection tube (supplied).
Close the lid, and centrifuge for 12000 x g, Room temperature, 00:00:15 ). Discard the flow-through.
On column DNAse I treatment
On column DNAse I treatment
15m
15m
Add 350 µL Buffer RW1 to the RNeasy spin column. Close the lid, and centrifuge for 12000 x g, 00:00:15 . Discard the flow-through.
Add the DNase I incubation mix (80 µL ) directly to the RNeasy spin column membrane, and incubate on the benchtop (20–30°C) for 00:15:00 .
Note
Be sure to add the DNase I incubation mix directly to the RNeasy spin column membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the spin column
15m
Add 350 µL Buffer RW1 to the RNeasy spin column. Close the lid gently, and spin 12000 x g, Room temperature, 00:00:15 . Discard the flow-through
Washing samples
Washing samples
Add 500 µL Buffer RPE to the RNeasy spin column. Close the lid, and spin for 12000 x g, Room temperature, 00:00:15 . Discard the flow-through
Repeat step 20 at least 4 times
Note
*This differs from the original protocol and you will need to order an additional bottle of RPE buffer to supplement what is supplied in the kit *
This is critical to ensure a yield of highly pure RNA, reflected in A260/A230 ratios >1.8. Otherwise, samples will likely be contaminated with residual guanidium salts and leaf pigments.
Transfer the column to a new 2 mL collection tube. Add 500 µL Buffer RPE to the RNeasy spin column. Close the lid, and spin for 12000 x g, 00:00:15 . Discard flow-through after inspecting the color.
Note
* This differs from the original protocol *
Samples are transferred to a new collection tube in this instance to allow you to see if the flow-through is colorless. If it is not, repeat the RPE washes until no coloration can be seen in the sample. Realistically this shouldn't be more than one additional spin, if your flow-through is still very green you probably have too much sample in the first place and should consider starting again.
Transfer the spin column to a fresh tube, spin for 12000 x g, 00:02:00
Note
This is to ensure removal of residual EtOH on the column, it is important to ensure high purity RNA samples for downstream analysis
Place the RNeasy spin column in a new nuclease-free 1.5 mL collection tube (supplied). Add 50 µL RNase-free water directly to the spin column membrane.
Close the lid, and spin for 12000 x g, Room temperature, 00:01:00 to elute the RNA.
Place samples on ice or store at -80 °C
Note
For best results, it is advisable to proceed directly to downstream applications.
e.g. check RNA amount and purity using a Nanodrop. A260/A280 ratio should be >2 and A260/A230 ratio should be >1.8.
Check RNA integrity using Qubit Fluorometer and perform cDNA synthesis