RNA extraction

RNA isolation from cells allows an analysis of the current cell activity at a given time, since only the genes that are also currently transcribed in the cell are present at the moment of isolation as RNA. Thus, the RNA isolation is an important technique in molecular biology and the isolated RNA may be used in various methods for the analysis of gene expression.

Special features when handling RNA

RNases (enzymes that catalyze the cleavage of RNA into smaller fragments) are very stable and may still be active even after autoclaving. RNA is very susceptible to degradation by RNases, whose natural function is to Mg2 - independent hydrolysis of phosphodiester bonds in the phosphate backbone of the RNA. Therefore, handling of RNA requires more care than working with the much more stable DNA. To avoid RNA degradation, RNA and RNases should be separated from each other at an early stage and to prevent introducing RNases from the environment into the sample. Therefore, disposable gloves should be worn, because RNases are produced by all organisms and, therefore, are also present in human sweat on the skin surface. In addition, it is useful to use a specific set of consumables ( tips, tip boxes, etc.) only for RNA experiments. In addition, specific RNase-free water should be used.

RNA isolation

As already mentioned, it is very important for the isolation of RNA, as early as possible to separate RNase of the RNA. All methods are based on the isolation of RNA, to lyse the cells in a chemical environment in which RNases are rapidly denatured. The RNA is then separated from the remaining cellular components. In this manner, total RNA. This total RNA can either use directly for further experiments (eg, Northern blot, reverse transcription into cDNA ), or it can be used as a starting material for the isolation of mRNA.

Method of Chomczynski and Sacchi

This method worked ( PEQLAB eg TRIzol Reagent ® from Invitrogen or TriFast ™ ) with a special reagent. Thus, it is possible to obtain according to a certain protocol RNA from cells or tissues. This method is based on the so-called single -step method of Chomczynski and Sacchi. Trizol containing guanidinium thiocyanate, which lyses the cells and simultaneously inactivates RNases and other enzymes. In addition, the reagent contains phenol, in which RNA triggers. By the addition of chloroform, followed by centrifugation, the phase separation occurs. Then the three phases can be seen. The upper aqueous phase containing RNA interphase DNA and the lower chloroform phase proteins. The RNA in the aqueous phase is then precipitated with isopropanol or ethanol. After two washing steps, RNA is dissolved in, for example, RNase-free water and is available for further applications.

" Nonidet P-40 " - method

This method is not suitable for tissue and serves for the extraction of mRNA from the cytosol. The advantage of this method is that the cell nuclei remain intact and therefore in addition the possibility to isolate DNA. The principle is based on the nonionic detergent Nonidet P-40, which is added to the cells and the DNA ( nuclei ) is deposited after centrifugation as a pellet. RNA, proteins and cell debris remain in solution. Subsequently, as in the single-step method, the insulation with phenol / chloroform.

Kit systems

As another possibility, the RNA isolation kit many systems from different companies and in numerous designs are available ( eg, RNeasy Mini Kit from Qiagen ® ). In these kit systems using small little columns that specifically bind RNA.

Concentration determination by spectrophotometry

With the spectrophotometric determination of the concentration measuring the optical density at λ = 260 nm ( OD260 ), the absorption maximum of nucleic acids (DNA, RNA), and λ = 280 nm ( OD280 ), the absorption maximum of proteins. If the sample is contaminated with genomic DNA or proteins, can be determined by the ratio of OD260 and OD280. For pure RNA, the ratio should be approximately 2.0. If the value is below the sample protein, genomic DNA, and / or aromatic substances, is contaminated. In this case, the RNA should be cleaned once again. Since an OD260 of 1 corresponds to this 40 g / ml RNA, the RNA concentration can be calculated with the following formula:

Concentration [ g / ml ] = OD260 × 40 ug / ml × dilution factor

Control of RNA integrity by agarose gel electrophoresis

With the aid of agarose gel electrophoresis of nucleic acids can be separated according to their size, wherein small fragments migrate faster than larger ones. The method is based on the migration properties of the nucleic acids that migrate through their negatively charged phosphate groups an electrical voltage applied toward the anode (positive pole). For an agarose gel is used, then by various dyes (eg, methylene blue) can be stained. Thus, the RNA can be made ​​visible and photographed. In intact RNA in the gel are two distinctly separate bands to detect the 28S and 18S ribosomal RNA bands. The 2:1 ratio of the fluorescence intensities of the 28S and 18S rRNA bands is a sign that the mRNA is not degraded. The 5S band is usually little or no see.

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