RNA isolation
RNA isolation is a crucial laboratory technique used to extract ribonucleic acid (RNA) from cells, allowing researchers to analyze gene expression and understand cellular processes. This method is necessary because all cells contain the same genetic information, but the expression of genes can vary greatly depending on environmental conditions or cell types. The process begins with cell lysis, which involves breaking open the cells in a way that prevents RNA degradation. This can be achieved through techniques such as freezing cells in liquid nitrogen or using strong denaturants.
Following lysis, it is essential to denature proteins and inactivate RNases—enzymes that degrade RNA. Various purification methods exist, including ultracentrifugation and column-based techniques, which separate RNA from other cellular components. After purification, RNA must be handled with care, often requiring storage at very low temperatures to maintain its integrity. Specific procedures may be employed to isolate RNA from particular cellular compartments or to remove unwanted RNA types, such as ribosomal RNA.
As advancements in genetic research continue, the ability to isolate intact RNA has become increasingly important, facilitating techniques like RT-PCR and microarray analysis. These methods enable scientists to investigate gene expression patterns across different tissues and conditions, contributing valuable insights into biological functions and disease mechanisms.
RNA isolation
SIGNIFICANCE: All cells in an organism or population of organisms of the same species contain the same (or nearly the same) set of genes. Therefore, understanding which genes are expressed under different conditions is critical to answering many questions in biology, including how cells differentiate into tissues, how cells respond to different environments, and which genes are expressed in tumor cells. The starting point for answering those questions is RNA isolation.
Cell Lysis
RNA isolation is a difficult proposition. RNA has a short life span in cells (as short as minutes in bacteria), and it is somewhat chemically unstable. In addition, enzymes that degrade RNA (RNases) are widespread in the environment, further complicating the task of separating intact RNA from other molecules in the cell.
The first step in RNA isolation is rapidly breaking open cells under conditions where RNA will not be degraded. One method involves freezing cells immediately in liquid nitrogen, then grinding the cells in liquid nitrogen in order to prevent any RNA degradation. Other methods involve lysing cells in the presence of strong protein denaturants so that any RNases present in the cell or the environment will be rapidly inactivated. The difficulty of the cell step depends substantially on the type of cell involved. Bacterial and fungal cells are typically much more difficult to break open than cells from mammals. As a consequence, it is often more difficult to isolate intact RNA from bacteria and fungi. A study found that a universal lysis buffer of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (βME) could overcome these difficulties.
Protein Denaturation and Further Purification
The next step in RNA isolation is to denature all proteins from the cell, to ensure that RNases will be inactive. In many cases, this is done at the same time as cell lysis. RNases are among the most resilient enzymes known, capable of being boiled or even autoclaved, yet retaining the ability to cleave RNA once they cool down. Consequently, the RNA next needs to be separated from RNases and other proteins to ensure that it will remain intact.
The separation of RNA from the rest of the macromolecules in the cell can be accomplished in a number of ways. One of the older methods for purifying RNA uses ultracentrifugation in very dense cesium chloride solutions. During high-speed centrifugation, these solutions create a gradient, with the greatest density at the bottom of the tube. RNA is the densest macromolecule in the cell, so it forms a pellet in the bottom of the ultracentrifuge tube. A newer technique for RNA purification involves the use of columns that bind RNA but not other macromolecules. The columns are washed to remove impurities, such as DNA and proteins, and then the RNA is eluted from the column matrix. A newer technique is based on the observation that, at an appropriate pH (level of acidity), RNA partitions into the water phase of a water-organic mixture. DNA and proteins either are retained at the boundary of the water-organic mixture or are dissolved in the organic phase.
Once the RNA is isolated, it needs to be handled carefully to ensure that it will not be degraded. Normally this involves resuspending the RNA in purified water, adding an alcohol solution, and storing it at −70 or −80 degrees Celsius (−94 or −112 degrees Fahrenheit). The purified RNA can then be used in a variety of techniques that help determine which genes are being transcribed in particular cells or tissues. These techniques include RT-PCR, Northern hybridization, analysis, and the construction of cDNA libraries.
Special RNA Isolation Procedures
In some cases, a geneticist wants to isolate only RNA from the cytoplasm of the cell, since RNA from the nucleus may be more heterogeneous. In this case, cells are lysed using a gentle detergent that disrupts the cytoplasmic membrane, without disturbing the nuclear membrane. Centrifugation is used to separate the nuclei from the cytoplasm and then the cytoplasmic RNA is further purified as described above.
For some procedures, such as RT-PCR, the RNA sometimes needs to be further purified to ensure that no contaminating DNA is present. In this case, the RNA sample may be treated with the I, which destroys DNA but leaves RNA intact.
For other procedures, like construction, the RNA is often purified to remove ribosomal RNA (rRNA), transfer RNA (tRNA), and other stable RNAs, since the majority of RNA in the cell (typically more than 90 percent) is and tRNA. In this case, the RNA solution is treated by incubating it with single-stranded DNA containing a chain of eighteen to twenty thymine nucleotides, either on a column or in solution. Messenger RNA (mRNA) from eukaryotes contains runs of twenty to two hundred adenine nucleotides that bind to the single-stranded DNA and allow the to be purified away from the stable RNAs.
Like most techniques in genetics, RNA isolation methods have improved greatly over the years. With advances in methods for studying such as microarray analysis, isolating intact RNA is a technique that is more critical than ever in the genetics laboratory.
Key terms
- cDNA librarya set of copies, or clones, of all or nearly all mRNA molecules produced by cells of an organism
- complementary DNA (cDNA)also called copy DNA, DNA that copies RNA molecules, made using the enzyme reverse transcriptase
- microarray analysisa method, requiring isolated RNA, that allows simultaneous determination of which of thousands of genes are transcribed (expressed) in cells
- reverse transcription polymerase chain reaction (RT-PCR)a technique, requiring isolated RNA, for quickly determining if a gene or a small set of genes are transcribed in a population of cells
- RNAribonucleic acid, the macromolecule in the cell that acts as an intermediary between the genetic information stored as DNA and the manifestation of that genetic information as proteins
- RNasesribonucleases, or cellular enzymes that catalyze the breakdown of RNA
Bibliography
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