As with any sample, when isolating nucleic acids, the state of the source material dictates the method of its disruption. Most of the biological material is easily to break down using chemicals or ultrasound, so there is no problem in getting the nucleic acids out of cells. The kits for general nucleic acid isolation rely on the maximum lysis of the starting material; without this crucial step, there will be no nucleic acids at the end of the isolation. But what method would you use for hard material like Arabidopsis seeds where the seeds are small, and the seed coat is tough? Or for yeast or insects that are encased in chitin?
It’s Cold in Here
Liquid nitrogen is the answer to disrupting most hard substances. Freezing in liquid nitrogen makes the material brittle and prone to shattering. While frozen, you can grind it with mortar and pestle (always wear gloves and safety glasses). Using quartz sand and a grind helper will improve the yield. More “high tech” solutions for larger seed volumes would be using a coffee grinder instead of mortar and pestle.
Remember: there is no kit that will provide you with good results if your starting material is poorly ground. Another source of disappointing results is improperly storing the material. Do not allow your ground seed powder to defrost until you are extracting nucleic acids. Keep it frozen at – 70 C or use immediately.
The next step in Arabidopsis seed nucleic acid extraction is resuspending the seed powder. If you are working with RNA, it’s essential to prevent material oxidation because RNA is much less stable than DNA (in fact, it’s notorious as being prone to degradation), and oxidation is the first step do RNA degradation. Add ?-mercaptoethanol or other antioxidant to the extraction buffer. The resuspended powder is used as a starting material for your manual or kit RNA extraction.
However, the problem with RNA extraction from seeds is often compounded between the incomplete seeds grinding or RNA degradation, and the interference of other compounds in the seeds with the extraction buffer. In essence, seeds are storage containers that need to: (1) be protected from being eaten by nucleases; and (2) contain all the energy necessary for the development of the emerging shoot. Therefore, depending on the plant species, seeds may contain phenolic compounds and be abundant in carbohydrates, respectively. These two survival traits make extracting RNA incredibly difficult.
For example, starch from Arabidopsis seeds causes sample solidification if you use guanidine isothiocyanate (GITC)-based RNA extraction buffers such as TRIzol and RNeasy kits. Starch can also interfere with RNA resuspension. Thus, a manual LiCl-Phenol method is more suitable for starch-rich seeds (1). LiCl-Phenol selectively precipitates RNA, leaving behind DNA, proteins, and carbohydrates. Depending on the size of the RNA of interest, you can vary the LiCl concentration and centrifugation time. Just be aware that this method may be unsuitable for recovery of the smallest RNA around 50 nucleotides. That may be an advantage for purifying your RNA probes, but if you are interested in miRNA, switch to the ethanol precipitation (2).
If your seeds contain a high level of phenol compounds (i.e. cotton seeds), use the hot borate method (2). Unlike the LiCl-Phenol method where the buffer is acidic, the extraction buffer for the hot borate method is alkaline. The buffer is supplemented with PVP-40, deoxycholate, and/or NP-40 as chaotropic agents (3).
All in all, if you are having a problem with nucleic acid extraction from seeds, first try improving the quality of your extract by ensuring complete seeds disintegration and thorough resuspension. If this doesn’t help, switch to manual extraction. Kits are time-saving shortcuts, but if they don’t work, nothing beats a little elbow grease.
- Rapid method for high-quality RNA isolation from seed endosperm containing high levels of starch. Li Z. and Trick H.N. BioTechniques (2018) 38:872-876
- A Modified Hot Borate Method Significantly Enhances the Yield of High-Quality RNA from Cotton (Gossypium hirsutum L.) Wan C.Y., WilkinsT.A. Analytical Biochemistry (1994) 223(1): Pages 7-12