RNA extraction of various plants, animals, and microorganisms, there is a manual in each kit. The experimenter simply followed the instructions. There is no need to prepare other reagents when using, which is very convenient and suitable for rapid extraction of RNA. RNA Extraction Kit has good integrity and high purity and can be used for molecular biology post-experiment. However, the better kits are more expensive, and the use of RNA Extraction kits can be determined according to their own experimental needs in the laboratory.
In molecular biology, extracting high-quality RNA is crucial for various studies, including gene expression analysis, functional genomics, and plant physiology research. However, plant tissues can present a unique challenge due to the presence of polysaccharides, secondary metabolites, and cell wall components that interfere with RNA isolation. To overcome these obstacles, plant RNA extraction kits have become essential tools for researchers.
Operation procedure
- Sample handling
- Plant tissue: Take fresh or frozen 100mg tissue at -70℃, grind in liquid nitrogen, add powder to 1ml lysate, and mix well.
- Animal tissue: Take fresh or frozen 100mg tissue at -70℃ and add 1ml lysate, homogenize with a tissue grinding pestle or homogenizer.
- Adherent cells: lysate lysate cells were directly added to the culture plate, and 1 ml lysate was added for every 106 cells. Mix well with a sampler.
- Cell suspension: Cell collection by centrifugation. Add 1 ml lysate for every 106 animal, plant, and yeast cells or every 107 bacterial cells.
- Blood treatment: Take 0.2-1ml fresh blood and add 3 times the volume of red blood cell lysate, mix it well, place it at room temperature for 10 minutes, and centrifuge it at 10000rpm for 1 minute. Discard the supernatant, if the precipitate contains red blood cells, you can add 2 times the volume of red blood cell lysate to repeat the cleavage step. After centrifugation, precipitate and add 1 ml lysate to mix well.
- The treated sample is placed at room temperature for 5 minutes so that the nucleic acid-protein complex is completely separated.
- Add 0.2ml chloroform to the homogenate sample, cover the tube, shake violently for 15 seconds, and leave it at room temperature for 3-5 minutes.
- Centrifuge at 12000rpm for 10 min at 2-8℃. The RNA is mainly in the colorless water phase in the upper layer, and transfers the water phase to the new tube, without attracting the precipitation.
- Pre-treatment of the adsorption column: Add 500ul washing liquid into the adsorption column, place it at room temperature for 2 minutes, centrifuge 2-8 at 12,000 rpm for 2 minutes, and discard the waste liquid.
- Add 200ul of anhydrous ethanol into the supernatant collected in step 4, mix well, add to the adsorption column, stand for 2 minutes, centrifuge at 2-8 12000rpm for 2min, and discard the waste liquid.
- Add 600ul bleach solution to the adsorption column (check whether anhydrous ethanol has been added before use), centrifuge 2-8 at 12,000 rpm for 2min, and discard the waste solution.
- Add 600ul bleach solution to the adsorption column, centrifuge 2-8 at 12,000 rpm for 2min, and discard the waste solution.
- Centrifuge at 12000rpm for 2 minutes, discard the collection tube and place the adsorption column at room temperature for several minutes to remove the residual bleaching liquid in the adsorption column.
- Put the adsorption column into a new tube, drip 50-100ul RNase-free ddH2O into the center of the membrane, leaving it at room temperature for 5 minutes, and centrifuge at 12000rpm for 2min So you get RNA.
Why is RNA Extraction Important?
Before diving into the details of plant RNA extraction kits, it’s essential to understand the importance of RNA extraction in plant research.
RNA (ribonucleic acid) serves as the intermediate molecule that translates genetic information from DNA into proteins. By studying RNA, researchers can gain insights into gene expression, which helps in understanding how plants respond to environmental stresses, pathogens, and developmental processes.
High-quality RNA is necessary for various downstream applications, such as:
- Quantitative PCR (qPCR): Quantifies gene expression levels.
- RNA Sequencing (RNA-Seq): Allows researchers to analyze the entire transcriptome.
- Northern Blotting: Used to detect specific RNA sequences.
- cDNA Library Construction: Essential for cloning and characterizing genes.
Thus, extracting pure, intact RNA is critical for generating reliable data in these experiments.
Challenges in Plant RNA Extraction
Plant RNA extraction poses some challenges compared to animal tissues due to the following factors:
- Cell Wall: Plant cells have rigid cell walls made of cellulose, which makes tissue lysis difficult.
- Polysaccharides: Co-extraction of polysaccharides can inhibit enzymatic reactions in downstream applications.
- Secondary Metabolites: Compounds like phenolics, tannins, and flavonoids can co-purify with RNA, leading to contamination and degradation.
To overcome these obstacles, specialized plant RNA extraction kits are designed to provide efficient protocols and reagents for isolating high-quality RNA from various plant species and tissues.
Plant RNA Extraction Kits: An Overview
Plant RNA extraction kits are commercially available solutions that simplify the process of isolating RNA from plant tissues. These kits typically contain optimized reagents, buffers, and purification columns or beads for efficient RNA isolation.
Key components of a plant RNA extraction kit include:
- Lysis Buffer: Breaks open the plant cells and releases RNA. It often contains guanidine thiocyanate or other chaotropic agents that help denature proteins and inhibit RNA-degrading enzymes (RNases).
- Precipitation Solution: Helps in separating RNA from DNA, proteins, and other contaminants.
- Binding Columns: Many kits use silica columns where RNA binds during the extraction process. This allows for easy washing and elution of pure RNA.
- Wash Buffers: Remove impurities such as proteins, polysaccharides, and secondary metabolites.
- Elution Buffer: Used to release the purified RNA from the binding column for downstream applications.
Step-by-Step Guide to Plant RNA Extraction Using a Kit
- Sample Collection and Preparation
Collect fresh plant tissue and snap-freeze it in liquid nitrogen. Alternatively, store the sample at -80°C until use. Frozen samples help in preventing RNA degradation.
Grind the frozen plant tissue to a fine powder using a mortar and pestle or a bead mill in the presence of liquid nitrogen. The finer the tissue, the better the RNA yield.
- Lysis of Plant Tissue
Add the appropriate amount of lysis buffer (as provided in the kit) to the ground tissue. The lysis buffer contains strong chaotropic agents that help dissolve cell walls and inactivate RNases.
Mix thoroughly by vortexing or shaking. Ensure that the plant tissue is completely lysed, and allow the mixture to sit for 5-10 minutes to ensure maximum disruption of the cells.
- Centrifugation and Removal of Debris
Centrifuge the lysate at high speed (10,000–12,000 x g) for 5–10 minutes. This will pellet the cell debris and unlysed material, leaving RNA in the supernatant.
Carefully transfer the clear supernatant to a new tube without disturbing the pellet. This step ensures that you are only working with the RNA-containing liquid phase.
- RNA Binding to the Silica Column
Depending on the kit, the next step typically involves adding ethanol or another binding solution to the supernatant. This helps the RNA bind to the silica membrane in the provided spin column.
Apply the mixture to the column and centrifuge according to the manufacturer’s instructions. The RNA will bind to the silica while contaminants pass through.
- Washing the RNA
After the RNA is bound to the column, wash the column several times with the provided wash buffers. These buffers help remove salts, polysaccharides, and other contaminants that can interfere with downstream applications.
Ensure that you centrifuge the column thoroughly after each wash to avoid any carryover of contaminants.
- RNA Elution
Elute the purified RNA from the column by adding the provided elution buffer or RNase-free water to the column. Centrifuge one final time to collect the RNA in a clean microcentrifuge tube.
Your eluted RNA is now ready for analysis or storage at -80°C.
Tips for Maximizing RNA Yield and Quality
- Use Fresh Tissue: Whenever possible, use fresh or properly frozen tissues. Degraded samples result in lower RNA yield and quality.
- Grind Thoroughly: Fine grinding of tissue is crucial for efficient RNA extraction. Poorly ground tissue can lead to incomplete lysis and lower yield.
- Keep Everything Cold: Work quickly with frozen tissue and keep tubes on ice to minimize RNA degradation.
- Check RNA Quality: Use a spectrophotometer (e.g., NanoDrop) to assess the purity of your RNA. A good-quality RNA sample should have an A260/A280 ratio of 1.8-2.1, indicating minimal protein contamination.
Troubleshooting Common Issues
- Low RNA Yield
- Cause: Incomplete tissue disruption or poor lysis.
- Solution: Ensure thorough grinding and complete mixing with the lysis buffer.
- RNA Degradation
- Cause: RNase contamination or improper handling.
- Solution: Use RNase-free reagents and equipment. Work quickly and keep samples cold to minimize degradation.
- High DNA Contamination
- Cause: Inefficient separation of RNA and DNA.
- Solution: Include an optional DNase treatment step to remove residual DNA.
- Co-extraction of Polysaccharides or Phenolics
- Cause: Polysaccharides or secondary metabolites co-purify with RNA.
- Solution: Use a specific RNA extraction kit for plant tissues rich in polysaccharides or phenolics, or add wash steps with a high-salt buffer.
Choosing the Right Plant RNA Extraction Kit
Several companies offer plant RNA extraction kits, and the kit choice depends on your plant species, tissue type, and downstream application. Popular brands include:
- Qiagen RNeasy Plant Mini Kit: Ideal for most plant tissues, offering reliable results with minimal polysaccharide contamination.
- Zymo Research Direct-zol RNA Miniprep: Suitable for fast extractions, compatible with TRIzol reagent.
- Sigma-Aldrich Spectrum Plant Total RNA Kit: Excellent for extracting RNA from challenging tissues like seeds and roots.
When selecting a kit, consider factors like ease of use, compatibility with your plant species, and the purity of RNA required for your experiments.
Conclusion
Plant RNA extraction may seem daunting, but with the right RNA extraction kit and proper techniques, it becomes a manageable and reproducible process. By following the steps outlined in this guide and choosing the appropriate kit, you can ensure that your RNA samples are of high quality and ready for downstream applications. Whether you’re performing gene expression analysis or RNA sequencing, high-quality RNA is the foundation of reliable, reproducible data in plant molecular biology.
