The Basics of RNA Extraction
Pure, high-quality RNA is necessary for real-time polymerase chain reaction (qPCR). While the general steps for isolating RNA are consistent between sample types, there are some key considerations when extracting RNA specifically from plants and leaves.
There are four main steps in isolating RNA:
- Disruption of cells or tissue
- Inactivation of endogenous ribonucleases (RNase)
- Denaturation of nucleoprotein complexes
- Removal of contaminating DNA and proteins
Plant samples can be very fibrous and contain high levels of polyphenols, polysaccharides and RNases. Methods for RNA extraction from plants must effectively break open the cells while maintaining the integrity of the target RNA.
Choose a Suitable Approach for Your Plant Sample
There are four main approaches for extracting and isolating RNA: organic extraction, spin filter formats, magnetic particles, and direct lysis.
Many commercial kits use spin filter formats to efficiently extract and isolate RNA. The steps involve homogenizing the sample, removing genomic DNA, capturing total RNA on a membrane filter, washing away contaminants, and eluting pure RNA.
Because plant samples, such as roots and seeds, can be particularly difficult to break open, it is important to select a kit that uses an effective disruption method. For instance, MP Bio’s FastRNA Win Kit contains its Lysing Matrix Z, a 2 mm specially stabilized zirconium oxide particle mix, that can efficiently lyse most plant samples.
Pro Tips for Troubleshooting Common Problems
|Common Problem||Possible Cause||Pro Tip|
|Low A260/A280 Values||RNA sample is diluted in water||Do not use RNase-free water to dilute the sample for measuring RNA purity. Use a neutral buffer such as 10 mM Tris/HCl, pH 7.0.|
|Protein contamination||Use less sample and/or repeat the washing step.|
|RNA sample is too dilute||Concentrate the sample.|
|Total RNA does not perform well in qPCR||Ethanol is present||Increase g-force or centrifugation time when drying the RNA Spin Filter.|
|Salt is present||Ensure that RNA Wash Buffer 1 and 2 are stored at room temperature. Check RNA Wash Buffer 1 and 2 for salt precipitates. If precipitates are present, dissolve them by careful warming.|
|DNA Contamination||Too much starting material||Reduce the amount of starting material. Perform DNase digestion of the eluate containing the total RNA.|
|Suboptimal homogenization of DNA binding carrier||Ensure the lysis solution is properly mixed.|
|Plant material contains DNA binding inhibitors||Reduce the amount of starting material and/or increase the volume of lysis solution. Perform DNase digestion of the eluate.|
|Clogged RNA Spin Filter||Insufficient homogenization of the plant sample||Increase homogenization time, centrifuge the sample and use the supernatant.|
|Too much starting material||Reduce the amount of starting material or increase the volume of lysis solution.|
|Lysate is too viscous due to too much DNA||Divide the sample into two aliquots, adjust the volume to 900 µL with lysis solutions, and continue with the protocol.|
|Poor RNA yield||Insufficient homogenization||Reduce the amount of starting material and/or increase homogenization time.|
|Incomplete elution||Prolong the incubation time with the elution buffer or repeat the elution step.|
|Alcohol not added to the wash buffers (kit specific)||Confirm wash buffers were prepared appropriately. Do not use denatured alcohol.|
|Degraded RNA||Inappropriate handling of starting material||Work quickly. Immediately store plant material at -80°C for delayed processing.|
|Lysis solution is lacking DTT or β-Mercaptoethanol||Confirm that the lysis solution is prepared appropriately.|
|RNase contamination||Establish a ribonuclease-free environment. Wear gloves and use sterile techniques throughout the process.|
Learn more tips on addressing challenges in plant research.