Feb 15, 2025

Public workspaceCTAB-Based RNA Extraction with Qiagen RNeasy Cleanup

DOI
dx.doi.org/10.17504/protocols.io.261gerk8wl47/v1
  • 1Cornell University
  • Puregene
Gonzalo Villarino
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.261gerk8wl47/v1
Protocol Citation: Gonzalo Villarino 2025. CTAB-Based RNA Extraction with Qiagen RNeasy Cleanup. protocols.io https://dx.doi.org/10.17504/protocols.io.261gerk8wl47/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 13, 2025
Last Modified: February 15, 2025
Protocol Integer ID: 120178
Keywords: RNA extraction, cannabis RNA isolation, CTAB method, Qiagen RNeasy cleanup, plant RNA purification, secondary metabolites removal, high-quality RNA isolation, polysaccharide-rich plant RNA, RNA-seq sample preparation, RT-qPCR RNA extraction, column clogging prevention, modified RNA extraction protocol, medicinal plant RNA isolation, chloroform:isoamyl alcohol RNA extraction, CTAB + silica column purification
Abstract
This protocol details a modified RNA extraction method that combines CTAB-based purification with Qiagen RNeasy column cleanup, specifically tailored for complex plant tissues like cannabis, which harbor high levels of secondary metabolites and polysaccharides. The CTAB extraction step effectively eliminates contaminants that could impede downstream applications, ensuring high RNA integrity. The following Qiagen cleanup step further improves RNA purity, resulting in high-quality RNA suitable for RT-qPCR, RNA sequencing, and various molecular biology applications. This protocol especially benefits researchers working with medicinal plants, recalcitrant species, or tissues rich in secondary metabolites. The comparative table provided in this protocol showcases key optimizations, contrasts this method with previously published techniques, and offers suggested adjustments for further refinements.


ABCD
AspectPureGene ProtocolPublished ProtocolsRecommended Adjustments
Alternative Precipitation MethodUses Sodium Acetate (NaOAc) precipitationSome protocols suggest Lithium Chloride (LiCl) precipitation for polysaccharide-rich samplesConsider testing LiCl precipitation if polysaccharide contamination remains
DNase Treatment for Cleaner RNAOn-Column DNase I treatment (15 min)Kiss et al. (2024) includes DNase treatment to remove gDNA contaminationKeep DNase treatment (15 min is sufficient)
RNA Integrity & YieldExpected yield: 2-91 µg/µl; Air-drying step before dissolving RNAKiss et al. (2024) reports RIN of 7.1–8.1 with similar workflowNo major changes needed
Polysaccharide & Polyphenol RemovalCTAB (2%), PVP-40 (2%), β-mercaptoethanol (2%)Kiss et al. (2024) uses β-mercaptoethanol (10% v/v)Increase β-mercaptoethanol to 10% v/v for better secondary metabolite removal
Phase Separation & RNA CleanupChloroform:Isoamyl Alcohol (24:1) + Qiagen RNeasy CleanupWang & Stegemann (2010) also integrate CTAB with silica column purificationNo major changes needed
This comparison reviews the CTAB + Qiagen RNA extraction protocol in relation to established plant RNA extraction methods. It assesses critical factors such as polysaccharide removal, phase separation, RNA integrity, precipitation techniques, and DNase treatment. Based on insights from the literature, suggested modifications are offered to enhance RNA yield and purity.












Attachments
Icon for Kiss et al. - 2024 - A modified CTAB method for the extraction of high-quality RNA from mono-and dicotyledonous plants ri.pdf
Kiss et al. - 2024 -...
1.2MB
Icon for Wang and Stegemann - 2010 - Extraction of high quality RNA from polysaccharide matrices using cetlytrimethylammonium bromide.pdf
Wang and Stegemann -...
1.3MB
Icon for Liyanage et al. - 2021 - A CTAB protocol for obtaining high-quality total RNA from cinnamon (Cinnamomum zeylanicum Blume).pdf
Liyanage et al. - 20...
1.1MB
Guidelines
  • This protocol combines CTAB-based RNA extraction for removing polysaccharides and secondary metabolites with Qiagen RNeasy column purification for high-purity RNA.
  • Work quickly but carefully to prevent RNA degradation—keep samples frozen in LN₂ whenever possible.
  • Use fresh reagents and pre-warm CTAB buffer to 65°C before starting.
  • Maintain an RNase-free environment by cleaning surfaces with RNaseZap and using filtered RNase-free pipette tips.
  • Do not overload columns—follow recommended sample volumes to avoid clogging.
Materials

1️⃣ Sample Collection & Preparation:

  • Cannabis flowers & scissors
  • Weigh boats & precision balance
  • Liquid nitrogen (LN₂) & Dewar flask
  • Ceramic mortar & pestle
  • Cryogenic-safe plastic containers

2️⃣ Safety & Waste Management:

  • Fume hood (for handling chloroform & β-mercaptoethanol)
  • Chemical waste collection bottle (HDPE or Glass, 1L or 2L, labeled: "Chloroform:Isoamyl Alcohol Waste – Hazardous Chemical Waste")
  • Biohazard waste bag (for contaminated disposables)
  • Nitrile gloves & safety goggles (for working with hazardous chemicals)
  • Absorbent spill pads (for chloroform & ethanol spills)
  • Flammable Storage Cabinet (100% Ethanol & chloroform:isoamyl alcohol are highly flammable)

3️⃣ RNA Extraction & Phase Separation:

  • RNA-Optimized CTAB Extraction Buffer (see Appendix)
  • Water bath (65°C, with floating tube racks)
  • Chloroform:Isoamyl Alcohol (24:1) – Must be used in fume hood
  • Sodium acetate (3 M, pH 5.2 for RNA precipitation)
  • Lithium chloride (LiCl, 8 M, for RNA precipitation)
  • Ethanol 100% & 75% Ethanol

4️⃣ Qiagen RNeasy Cleanup Kit & Buffers:

  • Qiagen RNeasy Plant Mini Kit (for Cleanup Step)
  • Buffer RLT (with β-ME added: 10 µL per 1 mL RLT)

5️⃣ Reagents for CTAB Buffer Preparation:

  • CTAB (for RNA extraction buffer preparation)
  • PVP-40 (removes polysaccharides and phenolic compounds)
  • NaCl (2 M, for CTAB buffer)
  • Tris-HCl (pH 8.0, 100 mM)
  • EDTA (25 mM, for chelation of RNase cofactors)
  • Spermidine (stabilizes RNA during extraction)
  • β-mercaptoethanol (2%, prevents oxidation of RNA by phenolics)
  • RNase-free water

6️⃣ Laboratory Equipment & Consumables:

  • Heat block
  • -80°C Freezer (For long-term storage of RNA samples)
  • -80°C Freezer (For reagents and RNA samples)
  • Water bath (65°C)
  • Centrifuge (capable of 12,000 x g, temperature-controlled)
  • Racks for Eppendorf 1.5mL tubes
  • Pipettes (P1000, P200, P10)
  • RNase-free pipette tips (P1000, P200, P10, filtered tips)
  • RNaseZap (for surface decontamination of workspace)
  • 2mL RNase-free tubes (for sample processing)
  • 1.5mL RNase-free tubes (for final RNA storage)

7️⃣ RNA Quality Control & Storage:

  • Qubit (for RNA quantification)
  • Tapestation ( for RNA integrity check)
  • -80

Safety warnings
  • Chloroform:Isoamyl Alcohol is hazardous—always work inside a fume hood and dispose of waste in a properly labeled chemical waste container.
  • β-Mercaptoethanol is toxic and volatile—handle only in a fume hood and wear gloves.
  • Ethanol and chloroform are flammable—keep away from open flames and use only in well-ventilated areas.
  • Liquid nitrogen (LN₂) can cause severe burns—handle with cryogenic gloves and never seal containers with LN₂ inside.
Before start
Prepare all reagents and equipment:
  • Pre-warm CTAB buffer to 65°C in a water bath.
  • Set up the centrifuge at 4°C and ensure it can reach 12,000 x g.
  • Chill spatulas in LN₂ before handling plant material.

Prepare working space:
  • Clean the bench with RNaseZap.
  • Set up a fume hood for handling chloroform:isoamyl alcohol and β-mercaptoethanol.
  • Label all tubes before starting to avoid confusion.

Ensure correct PPE (Personal Protective Equipment):
  • Wear gloves, safety goggles, and a face mask when handling hazardous chemicals.
  • Use cryogenic gloves when working with liquid nitrogen (LN₂).

Plan your workflow:
  • Keep samples frozen until the extraction step.
  • Work quickly to minimize RNA degradation.
  • Perform all centrifugation steps at the correct speed and temperature.
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.
Tissue Collection & Homogenization
Tissue Collection & Homogenization
Prepare fresh CTAB (see Appendix) before harvesting and grinding, keeping it at 65°C. Then, start the centrifuge (4°C).
Pre-chill spatulas in cryogenic-safe plastic containers with LN₂. Tare the calibrated precision balance before weighing samples.
Immediately flash-freeze in liquid nitrogen (LN2). Using a mortar and pestle, grind to a fine powder in LN2, ensuring the sample remains fully frozen during grinding!
Use a chilled spatula to transfer the powdered tissue into a 2mL RNase-free tube (ensure to use a 2mL tube).
RNA Extraction with CTAB
RNA Extraction with CTAB
Add 1 mL (use the P1000 pipette) of pre-heated CTAB buffer to the 2 mL RNase-free tube containing plant powder.
Vortex thoroughly for 1 minute
Incubate in a water bath at 65°C for 10 minutes, mixing occasionally (invert the tubes).
Add an equal volume (1mL) of chloroform:isoamyl alcohol 
Vortex for 30 seconds.
Incubate for 15 minutes at room temperature for optimal phase separation
Centrifuge at 12,000 x g for 10 minutes at 4°C
Transfer ~ 500 µL of the upper aqueous phase (RNA) to a fresh 1.5 mL RNase-free tube (do not disturb the interface between the aqueous vs. organic phase). Use the P200 pipette. The aqueous phase should be clear and colorless to pale yellow
RNA Precipitation: Sodium Acetate (NaOAc) + Ethanol (Standard Method)
RNA Precipitation: Sodium Acetate (NaOAc) + Ethanol (Standard Method)
Add 50 µL of 3M NaOAc and 1250 µL of 100% ethanol. Invert the tube 5–6 times to mix
Incubate at –20°C for 1 hour
Centrifuge at 12,000 x g for 20 min at 4°C. The RNA will be visible as a white pellet (bottom of the tube)
Discard the supernatant by removing the liquid slowly to discard the supernatant. Be careful not to touch the pellet with the pipette tip.
Wash the pellet with 1 mL of 75% ethanol. Invert the tube 5–6 times. The goal is to ensure the RNA pellet is well-washed without disrupting it. DO NOT VORTEX
Centrifuge at 12,000 x g for 5 minutes at 4°C. Pipette the ethanol out with a P200
Repeat Steps 18-19
Air-dry the RNA pellet for 10 minutes at RT to evaporate the residual ethanol
RNA Cleanup with Qiagen RNeasy Plant Mini Kit
RNA Cleanup with Qiagen RNeasy Plant Mini Kit
Pre-warm 400 µL Buffer RLT (with β-ME added) to 37°C in heat block
Dissolve RNA pellet in 400 µL Buffer RLT (with β-ME added). Pipette up & down gently until fully dissolved (If RNA does not fully dissolve, incubate at 37°C for 5 min and vortex briefly)
Pass the sample through a QIAshredder spin column (lilac) placed in a 2 mL collection tube. Centrifuge at ≥8000 x g for 2 min.
Carefully transfer the cleared lysate (~400 µL flow-through) to a new tube, avoiding any visible pellet at the bottom. Discard the QIAshredder column.
Add 200 µL of ethanol (0.5x volume, 96–100%) to the flow-through. Mix gently by pipetting up and down
Transfer the sample onto a pink RNeasy spin column (in a new 2 mL collection tube).
Centrifuge at ≥8000 x g for 15 sec. Discard the flow-through.
Add 700 µL Buffer RW1 to the spin column. Centrifuge at ≥8000 x g for 15 sec. Discard flow-through
Prepare DNase incubation mix: 10 μL DNase I (RNase-free)+ 70 μL Buffer RDD (from Qiagen RNase-Free DNase Set) + Mix gently (pipetting up and down)
Apply the DNase mix (80 μL) directly onto the membrane of the RNeasy spin column
Incubate at room temperature for 15 minutes to "kill" DNA
Add 350 µL Buffer RW1 to the spin column to wash out DNase.
Centrifuge at ≥8000 x g for 15 sec. Discard flow-through.
Add 500 µL Buffer RPE. Centrifuge at ≥8000 x g for 15 sec. Discard flow-through
Add 500 µL Buffer RPE again. Centrifuge at ≥8000 x g for 2 min(removes residual ethanol).
Place the RNeasy spin column in a new 2 mL collection tube. Centrifuge at full speed (≥10,000 rpm) for 1 min (ensures ethanol removal).
Transfer the RNeasy spin column to a new 1.5 mL RNase-free tube.
Add 30 µL of pre-warmed RNase-free water (37°C) directly onto the membrane. Let it sit for 1 min
Centrifuge at ≥8000 x g for 1 min to elute RNA.
Protocol references
A Modified CTAB Method for the Extraction of High-Quality RNA from Various Plant Species
* Summary: Optimized CTAB protocol for extracting RNA from woody and herbaceous plants. * Link: https://plantmethods.biomedcentral.com/articles/10.1186/s13007-024-01198-z


Extraction of High-Quality RNA from Polysaccharide Matrices Using CTAB

* Summary: Explains how CTAB efficiently isolates RNA from polysaccharide-rich plant tissues. * Link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2813910/


A CTAB Protocol for Obtaining High-Quality Total RNA from Cinnamon

* Summary: Optimized CTAB method for RNA extraction from different tissues of cinnamon plants. * Link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007690/



NOTE: these 3 PDFs are added in the "Description" section