Several non-column methods are established for RNA isolation in plant molecular biology and are preferred for cost-effectiveness, high yield, and compatibility with diverse and metabolite-rich tissues. Below is a semiformal summary of the main protocols and key steps involved.

Non-Column RNA Isolation Methods

Phenol-Chloroform Protocol

The phenol-chloroform method is widely used to remove proteins and polysaccharides from plant tissue, yielding purified RNA through sequential liquid-liquid extraction and precipitation steps. The plant tissue is ground in liquid nitrogen and homogenized in extraction buffer containing phenol and chloroform. The viscous mixture is thoroughly vortexed, followed by centrifugation to separate phases. The upper aqueous phase containing RNA is transferred to a new tube. RNA is precipitated with isopropanol or ethanol, washed with 70% ethanol, and resolubilized in RNase-free water. This protocol is particularly effective for plants with high secondary metabolite or polysaccharide content and does not require expensive spin columns or proprietary kits.

TRIzol Reagent Method

TRIzol reagent combines phenol and guanidine isothiocyanate, allowing rapid cell lysis, protein denaturation, and RNase inhibition. Plant material is homogenized in TRIzol, incubated, and mixed with chloroform. After centrifugation, the aqueous phase containing RNA is collected and precipitated with alcohol. This approach provides high yield and purity, especially for samples with challenging biochemical profiles. Modified protocols using additional phase separation steps or increased reagent volumes can be adapted for metabolite-rich tissues.

CTAB-LiCl Protocol

The CTAB-LiCl method is recommended for tissues rich in polysaccharides and polyphenols. CTAB (cetyltrimethylammonium bromide) buffer is used to lyse cells and form complexes with these interfering compounds. After homogenization, phenol-chloroform is added for protein removal and phase separation. RNA is selectively precipitated using LiCl, washed, and then dissolved for downstream use. This approach is robust and yields high-quality RNA even from woody, aromatic, or aquatic plants.

Modified SDS-LiCl Protocols

Recent advances include modified SDS-LiCl methods, which further improve reproducibility and RNA integrity for difficult plant tissues. SDS aids cell lysis, and LiCl allows selective precipitation of RNA while removing contaminants.

How RNA Isolated From Plant Samples

Key Steps

• Collection and Preservation: Fresh tissue is snap-frozen in liquid nitrogen and stored at –80°C to prevent RNA degradation.
• Tissue Homogenization: Frozen tissue is ground to a fine powder under liquid nitrogen to inactivate RNases and enable complete disruption of cell walls.
• Cell Lysis and Extraction: The powdered tissue is suspended in extraction buffer (phenol, CTAB, or TRIzol), which lyse cells and inhibit RNases.
• Phase Separation: Chloroform or phenol-chloroform is added, and following centrifugation, the RNA-containing aqueous phase is carefully collected.
• RNA Precipitation and Washing: RNA is precipitated from the supernatant using alcohol (isopropanol or ethanol), pelleted by centrifugation, washed with 70% ethanol, and dissolved in RNase-free water.
• Quality Assessment: The integrity and purity of RNA are typically assessed spectrophotometrically (A260/A280 ratios) and verified by gel electrophoresis.

Non-column protocols are especially useful for plants with high levels of secondary metabolites, polysaccharides, or polyphenols that interfere with column extraction. These methods remain the standard for cost-effective, flexible, and scalable RNA extraction in plant science.

Yes. There are indeed non-column (reagent-based) RNA isolation methods available for plants. These rely on liquid–liquid extraction (like phenol-guanidinium), proprietary reagents, or simpler buffers, and bypass the traditional silica-column purification. Here are some widely used options and specific kits:

• PR Reagent (Plant Total RNA Isolation Kit): A 3-step reagent system not requiring phenol extraction. Yields high-quality RNA (up to ~80 µg) in about two hours, suitable for RT-PCR, Northern blotting, and more. [GeneDireX]

• Genei Plant RNA Isolation Kit: Implements classic acid-phenol/chloroform extraction via SDS-based tissue lysis, followed by precipitation. Reliable for downstream cDNA synthesis and RT-PCR. [Genei]

• TRIpure Total RNA Extraction Reagent (TRIzol-equivalent): A Trizol-like reagent that efficiently isolates intact RNA from plant tissues, ideal for RT-PCR, qRT-PCR, RNA-seq, and expression studies. [Elk Biotechnology]

• Phenol-Free Plant RNA Isolation Kit (PlantMedia / Bioworld): A fast, phenol/chloroform-free protocol for DNA-free RNA, avoiding hazardous organic extraction. [Thermo Fisher Scientific]

• Acid Guanidinium Thiocyanate–Phenol–Chloroform Extraction (AGPC), e.g., TRIzol: A classic one-step liquid extraction method that offers high purity and recovery, skipping columns entirely. Particularly good for small RNAs (