RNA extraction

The RNA extraction includes biochemical methods for the extraction of RNA from cells . The isolated RNA can be used for numerous methods analysis of gene expression can be used. RNA extraction is a method of RNA purification .

RNases (enzymes that catalyze the cleavage of RNA into smaller fragments) have a high stability and can still be active after autoclaving. RNA is very susceptible to degradation by RNases, whose natural task is the Mg ²⁺ -independent hydrolysis of phosphodiester bonds in the phosphate backbone of the RNA. Therefore, handling RNA requires more care than working with the much more stable DNA. To avoid RNA degradation, RNA and RNases should be separated from one another at an early stage and the introduction of RNases from the environment into the sample should be prevented. Disposable gloves should therefore be worn because RNases are produced by all organisms and are therefore also present on the surface of the skin in human sweat. In addition, it makes sense to use a certain set of consumables (pipettes, pipette boxes, etc.) only for the RNA experiments. In addition, special RNase-free water should be used.

RNA isolation

Steps in RNA isolation

As already mentioned, when isolating RNA it is very important to separate RNases from the RNA as early as possible. All methods for isolating RNA are based on lysing the cells in a chemical environment in which RNases are rapidly denatured. The RNA is then separated from the other cellular components. In this way, total RNA is obtained. This total RNA can either be used directly for further experiments (e.g. Northern blot , reverse transcription in cDNA ), or it can be used as a starting substance for the isolation of mRNA .

After cell disruption, most RNA extractions are based on three different processes, two- phase extraction or precipitation , the latter possibly with additional selective adsorption onto an RNA-binding matrix. The RNA extraction procedures are similar to those used for DNA extraction . Some of the processes are also combined with one another. One of the methods is usually followed by a final isopropanol or ethanol precipitation .

Two-phase extraction

After cell disruption, RNA extraction can be carried out using a solution of phenol , chloroform and isoamyl alcohol in a volume ratio of 25: 24: 1. After addition to the cells, two phases are formed, an aqueous and an organic phase. The RNA collects in the aqueous phase. The aqueous phase is then subjected to isopropanol or ethanol precipitation of the RNA.

Single step method

RNA extraction by two-phase extraction and ethanol precipitation.

This method by Piotr Chomczynski and Nicoletta Sacchi uses a special reagent (e.g. Trizol , TRI Reagent , Trisure , TriFast , STAT-60 , RNAzol or DNAzol ). This makes it possible to lyse cells and at the same time to obtain RNA from cells or tissues. This process is based on the so-called single-step method according to Chomczynski and Sacchi. Trizol contains guanidinium thiocyanate , which lyses cells and at the same time inactivates RNases and other enzymes. The reagent also contains phenol , in which RNA dissolves. The phases are separated by adding chloroform and then centrifuging. Then three phases can be recognized. The upper aqueous phase contains RNA, the 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, the RNA is in z. B. RNase-free water and is available for other applications.

"Nonidet P-40" method

This method is not suitable for tissue and is used to obtain mRNA from the cytosol. The advantage of this method is that the cell nuclei remain intact and thus there is also the possibility of isolating DNA. The principle is based on the nonionic detergent Nonidet P-40 , which is added to the cells and the DNA (cell nuclei) settles as a pellet after centrifugation. RNA, proteins and cell debris remain in solution. As with the single-step method, isolation then takes place with phenol / chloroform.

adsorption

As a further option for RNA isolation, there are many kit systems available from various companies and in numerous designs. These kit systems use small columns that specifically bind RNA.

Catrimox-14 extraction

RNA and DNA form insoluble complexes with some cationic surfactants . During the Catrimox-14 extraction, the cells are lysed with the cationic surfactant Catrimox-14 ( tetradecyltrimethylammonium oxalate ) and guanidinium thiocyanate. After centrifugation and a washing step, extraction with phenol, chloroform and isoamyl alcohol (25: 24: 1 vol.) Takes place. The aqueous phase is then subjected to ethanol precipitation. The CTAB method for RNA or DNA extraction is carried out analogously , which, in the case of plant starting material, is more pure and less degraded than the single-step method.

Determination of concentration by photometry

When determining the concentration by spectrophotometry, the optical density is measured at λ = 260 nm (OD ₂₆₀ ), the absorption maximum of nucleic acids (DNA, RNA), and at λ = 280 nm (OD ₂₈₀ ), the absorption maximum of proteins . The quotient of OD ₂₆₀ and OD ₂₈₀ can be used to determine whether the sample is contaminated with genomic DNA or proteins . For pure RNA, the ratio should be around 2.0. If the value is below this, the sample is contaminated with protein, genomic DNA and / or aromatic substances. In this case the RNA should be purified again. Since an OD ₂₆₀ of 1 corresponds to 40 µg / ml RNA, the RNA concentration can be calculated using the following formula:

Concentration [µg / ml] = OD ₂₆₀  × 40 µg / ml × dilution factor

RNA integrity by agarose gel electrophoresis

RNA bands after gel electrophoresis
a: genomic DNA
b: 28S rRNA
c: 18S rRNA
d: 5S rRNA

With the help of agarose gel electrophoresis , nucleic acids can be separated according to their size, with small fragments migrating faster than larger ones. The method is based on the migration properties of nucleic acids, which migrate through their negatively charged phosphate groups in the direction of the anode (positive pole) when an electrical voltage is applied. An agarose gel is used for this, which can then be colored with various dyes (e.g. methylene blue ). This allows the RNA to be made visible and photographed. If the RNA is intact, two clearly separated bands, the 28S and 18S bands of ribosomal RNA, can be seen in the gel in this sample. The 2: 1 ratio of the fluorescence intensities of the 28S and 18S rRNA bands is a sign that the mRNA has not been degraded. The 5S band can usually hardly be seen or not at all.

history

→ Main article : “History” in the article DNA extraction

The history of RNA extraction is linked to the development of DNA extraction.

literature

• C Wagener, O Müller: Molecular oncology: origin, progression, clinical aspects . 3rd, completely updated and extended Ed. Thieme, Stuttgart [u. a.] 2009. ISBN 978-3131035134 .
• Friedrich Lottspeich , Haralabos Zorbas (Ed.): Bioanalytik. Spektrum Akademischer Verlag, Heidelberg et al. 1998, ISBN 3-8274-0041-4 .
• Cornel Mülhardt: The Experimenter: Molecular Biology / Genomics. Sixth edition. Spektrum Akademischer Verlag, Heidelberg 2008, ISBN 3-82-742036-9 .
• J. Sambrook , T. Maniatis , DW Russel: Molecular cloning: a laboratory manual. 3rd edition (2001), Cold Spring Harbor Laboratory Press. ISBN 0-87969-577-3 .

Web links

• RNase and DEPC Treatment: Fact or Laboratory Myth .

Individual evidence

1. ^ LV Madabusi, GJ Latham, BF Andruss: RNA extraction for arrays. In: Methods in enzymology. Volume 411, 2006, pp. 1-14, doi : 10.1016 / S0076-6879 (06) 11001-0 , PMID 16939782 .
2. ↑ Piotr Chomczynski, William Wilfinger, Karol Mackey: Single-Step Method of Total RNA Isolation by guanidine-phenol extraction . In: eLS (2013). doi : 10.1002 / 9780470015902.a0003799.pub2 .
3. ↑ SN Peirson, JN Butler: RNA extraction from mammalian tissues. In: Methods in molecular biology (Clifton, NJ). Volume 362, 2007, pp. 315-327, doi : 10.1007 / 978-1-59745-257-1_22 , PMID 17417019 .
4. ^ IE Jordon-Thaden, AS Chanderbali, MA Gitzendanner, DE Soltis: Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta. In: Applications in plant sciences. Volume 3, number 5, May 2015, p., Doi : 10.3732 / apps.1400105 , PMID 25995975 , PMC 4435465 (free full text).
5. ^ IM Bird: Extraction of RNA from cells and tissue. In: Methods in molecular medicine. Volume 108, 2005, pp. 139-148, PMID 16028681 .
6. ↑ P. Chomczynski, N. Sacchi: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. In: Anal Biochem . (1987) 162 (1): 156-9. PMID 2440339 .
7. ↑ SK Dutta, AS Jones, M. Stacey: The separation of desoxypentosenucleic acids and pentosenucleic acids. In: Biochimica et Biophysica Acta . Volume 10, Number 4, April 1953, pp. 613-622, PMID 13059025 .
8. ^ CE Dahle, DE Macfarlane: Isolation of RNA from cells in culture using Catrimox-14 cationic surfactant. In: BioTechniques. Volume 15, Number 6, December 1993, pp. 1102-1105, PMID 8292344 .
9. ^ CE Dahle, DE Macfarlane: Isolating RNA with the cationic surfactant, Catrimox-14. In: Methods in molecular biology (Clifton, NJ). Volume 86, 1998, pp. 19-21, doi : 10.1385 / 0-89603-494-1: 19 , PMID 9664447 .
10. ↑ Yolanda M. Camacho-Villasana, Neftali Ochoa-Alejo, Linda Walling, Elizabeth A. Bray: An improved method for isolating RNA from dehydrated and nondehydrated chili pepper (Capsicum annuum L.) tiss plantues. In: Plant Molecular Biology Reporter. 20, 2002, p. 407, doi : 10.1007 / BF02772128 .
11. ↑ K. Shahrokhabadi, RT Afshari, H. Alizade, JT Afshari, GR Javadi: Compared Two Methods for Isolating RNA from Freezing and Nonfreezing Bread Wheat (Triticum aestivum L.) Plant Tissues. In: Asian Journal of Plant Sciences. 7, 2008, p. 505, doi : 10.3923 / ajps.2008.505.509 .