A tool for polysaccharide and polyphenol plant RNA extraction
Many literature reports that many plants have hindered their research progress in molecular biology due to the failure to effectively isolate and purify RNA in their tissues. For example, Northern hybridization analysis, in vitro translation analysis or cDNA library construction, RT-PCR and differential display analysis and other studies require high-quality RNA. Therefore, the extraction of RNA with high purity and good integrity is the key to the smooth progress of the above research.
Common difficulties in RNA extraction
Studies have shown that the reason why it is difficult for some plant tissues to extract high-quality RNA is related to some of the rich ingredients in these plant tissues. Common ones are phenolic compounds, polysaccharides, and some undetermined secondary metabolites. In addition, There are tissues rich in higher activity RNase. In intact cells, these substances are separated from nucleic acids. Once the cells are broken, these substances will interact with RNA.
Many plant tissues, especially fruits (such as apples, cherries, plums, grapes, etc.) and trees are rich in phenolic compounds. As the plants grow, the content of phenols will increase, so young plants are the best extraction material. In addition, the content of polyphenols in the needles of coniferous plants is much higher than the leaves of deciduous plants.
After the plant material is crushed in the early stage, phenols will be released, and the treatment liquid will become brown after oxidation (see the figure below), and it will deepen with the increase of the degree of oxidation. This phenomenon is called the browning effect (browning effect) ). Oxidized phenolic compounds (such as quinones) can irreversibly bind to RNA, resulting in loss of RNA activity. When extracted with phenol and chloroform, RNA will also be lost or form insoluble complexes, which will affect RNA separation and purification. Newbury et al also found that there is no direct correlation between the ease of RNA extraction and the total amount of phenolic compounds in the material, but with the so-called "condensed tannin" that is polymerized polyhydroxyflavonols (such as proanthocyanidins) )related.
Polysaccharides are another common problem in plant RNA extraction. Plant tissue is often rich in polysaccharides, and many physical and chemical properties of polysaccharides are very similar to RNA, so it is difficult to separate them.
If polysaccharides are to be removed, RNA will also be taken away randomly, resulting in a reduction in RNA yield. When precipitating RNA, gelatinous precipitates of polysaccharides are often produced. Such precipitates are difficult to dissolve in water or produce a viscous shape after dissolution. Solution, so it is also difficult to effectively separate it from RNA. In addition, polysaccharides can inhibit the activity of many enzymes, so RNA samples contaminated with polysaccharides cannot be used for further molecular biology research. In the conventional method, some polysaccharides can be partially removed by SDS-guanidine hydrochloride treatment; in the presence of high concentration Na + or K + ions, some polysaccharides can also be removed by phenol and chloroform extraction; in addition, some of the RNA can also be precipitated by LiCl The polysaccharide remains in the supernatant. But even through these steps, quite a lot of polysaccharides and RNA will be found mixed together, so more effective methods are needed to solve the problem of polysaccharide contamination during the isolation and purification of plant RNA.
Interference with secondary metabolites
Many higher plant tissues, especially mature tissues, can produce certain water-soluble secondary metabolites. These secondary metabolites are easily combined with RNA and extracted together with RNA to hinder the separation of biologically active RNA. Since it is impossible to determine exactly what these secondary products are, there is currently no special method to completely solve this problem.
Secondary metabolite production process
Plant tissues, especially higher plant tissues, have complex and diverse components, so the extraction of plant tissue RNA is much more difficult than other biological materials. Therefore, it is really difficult to solve all the problems in the extraction of plant tissues with a single reagent. Therefore, it requires a dedicated researcher or reagent manufacturer to invest a lot of energy and financial resources to carry out in-depth research, and then develop a convenient and practical reagent Box comes.
Throughout the international RNA extraction kits, such as the common TRIzol, and some well-known brand RNA extraction kits, it is difficult to extract high-quality RNA from polysaccharide and polyphenol plants.
The main components of Trizol are guanidinium isothiocyanate and phenol, and there is no special reagent for removing polysaccharides and polyphenols, so most plants with polysaccharides and polyphenols cannot be successfully extracted, even if added such as 2-mercaptoethanol, dithiothreitol ( DTT) or cysteine â€‹â€‹to prevent phenol oxidation reagents are also difficult to extract. The main lysate of the RNeasy Plant Mini Kit is guanidine hydrochloride or guanidinium isothiocyanate. Some companies have added reagents such as PVP40 to bind polyphenols after improvement, and they have not been able to obtain satisfactory results.
Biotek has accumulated rich practical experience in RNA extraction and developed a universal plant total RNA rapid extraction kit (article number: RP3301 or RP3302), so that customers do not need to check a lot of related information, and do not need to spend a lot of time And work hard to find a kit for extracting suitable plant samples! In more than one hundred polysaccharide and polyphenol plants tested by Biotech, high-quality RNA was extracted without exception, including cotton, apple, grape, strawberry, banana, longan, lychee, tomato fruit, eggplant, pine needle, poplar Trees, Arabidopsis seeds, jackfruit, horseshoe gold, bluegrass, tall fescue, spruce, pine, tilia, rowan tree, birch, beech, begonia flowers, maple maple, violet, chrysanthemum, clove, rose, Geranium, cyclamen, chrysanthemum, morning glory, purple pine cone, coleus, poinsettia, oleander, weeping ficus, etc.
The features of the universal plant total RNA rapid extraction kit are as follows:
l High-quality imported adsorption membrane greatly reduces the difference in adsorption capacity between columns caused by other imported or domestic adsorption membranes, ensuring the repeatability of the experiment
l The unique combined extraction liquid has stable performance and high purity, which can greatly improve the binding efficiency, thus laying a good foundation for the yield of RNA
l Centrifugal column type is simple and fast, no need to use traditional isopropanol precipitation and ethanol washing, simplify the operation steps and improve the experimental efficiency. At the same time, it can avoid the problem that the extract is dry and not easy to dissolve.
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