Feb 14, 2025
Protocol: RNeasy Plant Mini Kit for RNA Extraction
- 1Cornell University
- Puregene
Protocol Citation: Gonzalo Villarino 2025. Protocol: RNeasy Plant Mini Kit for RNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk9yqdv5r/v1
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: February 14, 2025
Last Modified: February 14, 2025
Protocol Integer ID: 120297
Abstract
This protocol describes the RNA extraction process from cannabis flowers using the Qiagen RNeasy Plant Mini Kit. Given the lack of a standardized RNA extraction method for cannabis, this protocol provides optimized steps to ensure high RNA yield and integrity by minimizing degradation. The workflow includes sample collection in the greenhouse (GH), flash-freezing in liquid nitrogen (LN2), mechanical disruption, and column-based RNA purification. Pre-warmed RNase-free water at 37°C is used to maximize RNA elution efficiency.
Attachments
Guidelines
- Work quickly to prevent RNA degradation.
- Keep all tools and surfaces RNase-free by cleaning them with RNaseZap or 70% ethanol.
- Use fresh gloves and sterile tools for each sample.
- Flash-freeze all plant material immediately after liquid nitrogen or dry ice collection.
- Keep sample tubes and reagents cold during processing unless otherwise stated.
- Ensure ethanol (96-100%) is freshly prepared before use.
Safety warnings
- Do not allow tissue to thaw at any point before lysis.
- Buffer RLT and RW1 contain guanidine salts—avoid using with bleach or acid-based disinfectants.
- Ethanol is flammable—keep away from flames and heat sources.
- Wear protective equipment (gloves, lab coat, goggles) when handling β-mercaptoethanol.
- Always use an RNA-dedicated pipette and work in an RNase-free area.
Before start
Equipment:
- Portable analytical balance
- Liquid nitrogen (LN2) dewar
- Mortar & pestle (pre-chilled)
- Microcentrifuge (capable of ≥10,000 rpm)
- Pipettes (2 µL – 1000 µL) and RNase-free pipette tips
- Heat block set at 37°C (for pre-warming elution buffer)
Reagents & Consumables:
- Qiagen RNeasy Plant Mini Kit
- RNase-free water
- Buffer RLT (with β-mercaptoethanol, if needed)
- Buffer RW1
- Buffer RPE (ensure ethanol is added before use)
- Ethanol (96–100%)
- Sterile scissors & tweezers
- RNase-free 50 mL Falcon tubes (for sample freezing)
- RNase-free 2 mL & 1.5 mL microcentrifuge tubes
- QIAshredder spin columns (lilac)
- RNeasy spin columns (pink)
Preparation Before Going to the Greenhouse (GH)
Preparation Before Going to the Greenhouse (GH)
Set up a portable analytical balance in the GH
Label one tube per cultivar. Use 50 mL Falcon tubes
Bring RNase-free gloves and sterile tools (scissors, tweezers).
Go to the greenhouse and collect fresh cannabis flowers
Go to the greenhouse and collect fresh cannabis flowers
Cut fresh flowers using sterile scissors (one sample at a time)
Place the weighed ~100 mg sample into a labeled 50 mL Falcon tube. Poke some holes in lid of Falcon tube.
Flash-freeze the plant material (in 50 mL Falcon tube) immediately in liquid nitrogen
Go to lab
Go to lab
1m
1m
Grind the frozen material thoroughly with a mortar and pestle while keeping it in liquid nitrogen.
1m
Transfer the finely powdered material to an RNase-free, pre-chilled 2 mL microcentrifuge tube (not supplied)
RNA Extraction Protocol
RNA Extraction Protocol
Add 450 µL Buffer RLT to the 2 mL with the plant material. Vortex (1 minute) vigorously for complete lysis. Make sure the sample looks homogenous (no visible chunks or fibrous material)
Transfer ALL the lysate (~450 µL) from the 2 mL tube to a QIAshredder spin column (lilac) placed in a 2 ml collection tube. Centrifuge for 2 min at full speed.
Transfer the supernatant (~400 µL) of the flow-through to a new RNase-free 1.5 mL microcentrifuge tube (not supplied) without disturbing the cell debris pellet in the collection tube. Use only this supernatant in subsequent steps. Discard only the QIAshredder spin column.
Add ~200 µL (0.5 volume of 100% ethanol ) to the cleared lysate and mix immediately by pipetting (DO NOT VORTEX). Do not centrifuge. Proceed immediately to step 13.
Transfer the sample (~600 µl), including any precipitate that may have formed, to an RNeasy spin column (pink) placed in a 2 ml collection tube (supplied).
Close the lid and centrifuge for 15 seconds at ≥8000 x g (≥10,000 rpm). Discard the flow-through
Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid and centrifuge for 15 seconds at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.
Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid and centrifuge for 15 seconds at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.
Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid and centrifuge for 2 min at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane.
Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow-through. Close the lid and centrifuge at full speed for 1 min.
Pre-warming the RNase-free water to 37°C in heat block
Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30 µl RNAse-free water directly to the spin column membrane. Let it sit for 1 minute at room temperature. Close the lid. Centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA.
Protocol references
Qiagen RNeasy Plant Mini Kit Handbook
- Qiagen (2023). RNeasy Mini Handbook. Qiagen GmbH, Hilden, Germany.