RNA purification

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The RNA-purification (also RNA preparation, RNA isolation ) includes biochemical methods for the separation of RNA from a mixture or a solution containing a plurality of biomolecules.

Cell disruption

An organism's RNA can be isolated in several ways. Most of the methods begin with a concentration of the cells by centrifugation and a suitable cell disruption of the cell precipitate for the respective group . So z. B. in plant , fungal or bacterial cells, which in comparison to animal cells, mycoplasmas and some archaea species have a cell wall in addition to the cell membrane , usually additional enzymatic (e.g. lysozyme in bacteria or proteinase K for proteolysis ) or mechanical grinding steps ( blender ) respectively.

Digestion buffer

By adding strong surfactants such as 1% (m / V) sodium lauryl sulfate or N- lauryl sarcosine to the digestion buffer (synonymous with lysis buffer ), the cellular biomembranes (e.g. cell membrane , cell nucleus membrane , mitochondrial membrane) can be dissolved, but the RNA is not separated different cell organelles more possible. With chaotropes such as 8 molar urea or 6 molar guanidinium thiocyanate contained RNases can be denatured , and due to the high tonicity , the biomembranes sometimes tear . RNA from the cell nucleus, cytosol , mitochondria or chloroplasts , on the other hand, can be removed by hypotonic lysis with a digestion buffer of low ionic strength (1 mM buffered) and the comparatively mild surfactant Nonidet P-40 (0.05% m / V) and subsequent cell fractionation with retention of the Membranes of the cell organelles are separated. The cell membranes are partially loosened and the cytosolic RNA and the intact cell organelles are released from the cells. RNA with a poly-A tail can be separated from other RNA by affinity chromatography with an oligo-dT column . After cell disruption, the homogenate is usually clarified by filtration or centrifugation.

Rnoses

Since RNA is broken down comparatively quickly by cellular RNases and has a relatively short biological half-life , the extractions are carried out quickly at around 4 ° C (on ice). The solutions used are also made up with DEPC- treated, RNase-free water. With some solutions, the DEPC treatment can only be carried out with the finished solution, but not in the presence of TRIS or thiols . Often, the digestion buffer is also a thiol such. B. 10 millimolar mercaptoethanol or dithiothreitol added to inactivate RNases by cleaving their disulfide bridges . In some cases, RNase inhibitors such as RNasin or vanadyl ribonucleoside complexes (VDR) can be added. Protein-based RNase inhibitors also exist. The reagents and consumables used should be RNase-free in order to reduce the undesired degradation of the RNA. Glass containers are baked in the oven for at least 2 hours at 200 ° C over dry heat in order to inactivate RNases. Careful work can avoid contamination of the sample with RNases from the experimenter's skin and clothing. Purified RNA, which will be required for experiments in the next week, can be frozen at −20 ° C, while the longer-term storage of the isolated RNA in aqueous solution or the starting material usually takes place at −80 ° C. Sometimes RNA is stored in stabilized formamide .

Properties of RNA

RNA is a polar biopolymer with a relatively high molar mass , which is why it precipitates in a non-polar environment due to the reduced hydrate shell and the resulting reduction in its solubility . In addition, due to the ribose phosphate backbone with negative charges proportional to the chain length, RNA is insoluble in acidic, aqueous solutions. At low pH values , the phosphate groups and thus the negative charges of the RNA are saturated with protons , which also reduces the size of the hydration shell. RNA is a little more polar and water-soluble than DNA , as it has one more hydroxyl group per nucleotide . Compared to DNA, RNA is chemically more labile, especially at basic pH values ​​and at elevated temperatures (> 65 ° C). At high ionic strength, slightly acidic pH and low temperature, RNA more readily forms double strands , while the specificity of base pairing increases at low ionic strength, basic pH and higher temperature. At high ionic strength, the mutual repulsion of the negatively charged phosphate groups within the RNA decreases. At lower pH values, the phosphate groups are partially saturated with protons, which reduces the repulsion. The formation of an RNA double strand with complementary sequences is more stable than an RNA-DNA hybrid double strand or a DNA double strand, which is also reflected in the higher specificity of the base pairing of RNA compared to DNA under suitable conditions. The procedures for RNA purification are similar to those for DNA extraction due to the similarity of the two molecules.

Separation process

In the SEC, large particles such as RNA, RNA and polysaccharides elute before small ones (e.g. proteins , metabolites )

RNA extraction

A series of extractions and precipitations is probably the most commonly used procedure.

Chromatography

RNA can be separated by size exclusion chromatography (SEC) based on its hydrodynamic volume . RNA can also be separated by anion exchange chromatography .

sedimentation

By means of an equilibrium density gradient centrifugation in a cesium chloride or cesium trifluoroacetate gradient, RNA can be separated on the basis of its sedimentation constant .

The cells can be disrupted with urea (6 M ) and lithium chloride (3 M), followed by density gradient centrifugation with a sucrose gradient.

Using pulldown assays , RNA molecules are adsorbed on a matrix based on their affinity and isolated based on the properties of the matrix.

Electrophoresis

After another previous purification by agarose gel electrophoresis or by capillary electrophoresis, the RNA can be separated according to its electrical charge and hydrodynamic volume, both of which depend on the chain length and thus on the molar mass. The RNA can then be isolated by gel extraction of a specific band in the gel.

Filtration

In a series of microfiltration and several ultrafiltration , samples are also separated according to their hydrodynamic volume.

literature

Individual evidence

  1. ↑ a b c d e f g Robert E. Farrell, Jr .: RNA Methodologies. Academic Press, 2010, ISBN 978-0-080-45476-4 . Pp. 67-113.
  2. ^ Paul A. Krieg: A Laboratory Guide to RNA. John Wiley & Sons, 1996, ISBN 978-0-471-12536-5 .
  3. ↑ a b c d e f Gerard Meurant: RNA Methodologies. Academic Press, 2012, ISBN 978-0-323-13779-9 . Pp. 47-112.
  4. ↑ JM Chirgwin, AE Przybyla, RJ MacDonald, WJ Rutter: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. In: Biochemistry. Volume 18, Number 24, November 1979, pp. 5294-5299, PMID 518835 .
  5. ↑ K. Mommaerts, I. Sanchez, F. Betsou, W. Mathieson: Replacing β-mercaptoethanol in RNA extractions. In: Analytical biochemistry. Volume 479, June 2015, pp. 51-53, doi : 10.1016 / j.ab.2015.03.027 , PMID 25841674 .
  6. ↑ SE Zale, AM Klibanov: Why does ribonuclease irreversibly inactivate at High Temperatures? In: Biochemistry. Volume 25, Number 19, September 1986, pp. 5432-5444, PMID 3778869 .
  7. ^ NC Nicolaides, CJ Stoeckert: A simple, efficient method for the separate isolation of RNA and DNA from the same cells. In: BioTechniques. Volume 8, Number 2, February 1990, pp. 154-156, PMID 1690560 .
  8. ↑ P. Blackburn, G. Wilson, S. Moore: Ribonuclease inhibitor from human placenta. Purification and properties. In: The Journal of biological chemistry. Volume 252, Number 16, August 1977, pp. 5904-5910, PMID 560377 .
  9. ^ NR Murphy, SS Leinbach, RJ Hellwig: A potent, cost-effective RNase inhibitor. In: BioTechniques. Volume 18, Number 6, June 1995, pp. 1068-1073, PMID 7546711 .
  10. ↑ RE Kingston, P. Chomczynski, N. Sacchi: guanidines methods for total RNA preparation. In: Current protocols in molecular biology / edited by Frederick M. Ausubel .. [et al.]. Chapter 4 May 2001, S. Unit4.2, doi : 10.1002 / 0471142727.mb0402s36 , PMID 18265236 .
  11. ↑ C. Auffray, F. Rougeon: Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. In: European Journal of Biochemistry / FEBS. Volume 107, Number 2, June 1980, pp. 303-314, PMID 6772444 .