This article summarizes commonly used methods and kits for RNA extraction, presents the results of a Labome survey of randomized formal articles on RNA extraction and purification, and discusses recent comparative studies and two more popular extraction kits. , TRIzol and RNeasy.
DNA extraction methods cannot be applied directly to RNA as RNA is structurally very different from DNA. RNA is single-stranded, while DNA is mostly double-stranded. It is often difficult to isolate intact RNA. RNases, a group of enzymes that break down RNA molecules, are abundant in the environment, including on hands and on surfaces, and RNases are difficult to remove or destroy completely.
Therefore, RNA isolation requires the careful handling of samples and good aseptic techniques. It is important to use only RNase-free solutions during extraction, as well as pipette tips and RNase-free glassware.
RNA storage and stability
Many researchers use RNAlater solutions from Thermo Fisher and QIAGEN during RNA isolation, both to stabilize cellular RNA in tissue samples and to stabilize the final purified RNA. Environmental storage of tissue samples in RNAlater preserves RNA integrity in a manner similar to low-temperature storage.
RNAlater is based on the inhibition of RNAs by sulfate salts such as ammonium sulfate at a specific pH. The quality of the RNA can be checked by agarose gel electrophoresis. For long-term storage, RNA should be kept at -80 ° C in single-use aliquots, while ethanol precipitated from RNA can be stored at -20 ° C.
Determination of the quality and quantity of RNA
The quality of the RNA can be determined by examining the absorption ratio at 260 nm and 280 nm with UV spectrophotometry. For high-quality RNA, the A260 / A280 ratio should be in the range of 1.9–2.1. RNA can be quantified by measuring absorption at 260 nm, where 1 absorbance unit equals 40 µg / ml, at a pH of approximately 7.5. The validity of this relationship as a measure of RNA purity was questioned.
Furthermore, the quality of the total RNA preparations should be examined by electrophoresis, where the 18S and 28S RNA bands should be very prominent, with the 28S RNA band approximately twice as intense as the 18S band. Qubit Assays, provided by ThermoFisher Scientific, can also quantify RNA samples and assess their quality.
Deming et al examined the quality of RNA preparations from postmortem frozen human brain parietal lobe tissues with the RNA Peak 6000 assay using the Bioanalyzer 2100 from Agilent Technologies. Garrett-Bakelman FE et al used the Agilent RNA Nano Kit on an Agilent Bioanalyzer 2100 to examine the quality of RNA preparations from NASA twin astronaut blood samples.
Plant RNA extraction
RNA extraction methods must be tailored to the organism from which the RNA is extracted. Plants pose additional challenges due to the presence of secondary metabolites, polyphenols, and polysaccharides. Chan et al demonstrated that the addition of polyvinylpyrrolidone (PVP) to the extraction buffer helps to remove phenolic compounds and polysaccharides from mangosteen leaves and flowers.
Birtic et al used an optimized concentration of PVP, together with DNase and ethanol precipitation, to isolate high-quality RNA from seeds of five plant species, while Xie et al developed modified RNA extraction methods to obtain High-quality RNA from cotton. roots. Tattersall et al evaluated 15 methods/kits and found that tris-lithium chloride and RNeasy Midi + polyethene glycol gave the best quality RNA from grapevine leaves; published their results in the American Journal of Enology and Viticulture.
Commercially available RNA isolation kits designed for plants are available, such as the Spectrum Plant Total RNA Kit from MilliporeSigma and the RNeasy Plant Kits from QIAGEN and the Plant Kit. Macherey-Nagel NucleoSpin RNA.