by Yasemin Cole
Fun rating: 3/5
Difficulty rating: 2/5
What is the general purpose? To separate one of the smallest units of genetic information called ribonucleic acid (RNA) from cellular material.
Why do we use it? We use RNA extraction to study the quantity of RNA in a sample (e.g. the detection of COVID RNA molecules in a nasal swab). Using a technique called qPCR, we can study how much protein is being made by cells. For example, we may want to know the quantity of RNA present in the blood of someone infected with SARS-CoV-2. If RNA is present, we assume that the virus is replicating, and we can detect the relative amounts of RNA in the blood sample. We can also study it directly by looking at the sequence of RNA (similar to DNA with technologies such as next-generation sequencing).
How does it work? You may have heard of the phrase “DNA is the master blueprint for life.” What this doesn’t take into account is the important role of RNA. DNA consists of a combination of the nucleotides adenine, cytosine, guanine and thymine. RNA is similar to DNA except for one base that codes for uracil instead of thymine. RNA is a messenger between DNA and protein (see image above) and is used to store the information held by DNA to be eventually translated into proteins. So how can we isolate RNA from DNA and other components of the cell?
Step 1: Cell Lysis
The first step involves physically and/or chemically breaking the outer membrane of the cell such that the cellular contents will spill out. This process is called lysis and is also used in the DNA extraction process.
Step 2: RNA Isolation
Using the chemicals phenol and chloroform, the contents of the cell are split into a water-based (aqueous) phase located at the top of the tube and a water-repelling (organic) phase located at the bottom of the tube. Water-loving molecules such as RNA will be attracted to move into the lighter aqueous phase, whereas water-repelling molecules will be attracted to move into the heavier organic phase. As illustrated in the image above, the aqueous phase at the top of the tube is then moved into a separate tube for further processing.
Step 3: RNA Purification and Precipitation
To separate the RNA from other molecules in the aqueous phase, a combination of washes with ethanol (alcohol) and salts forces the RNA to solidify or precipitate out of the solution. A filter may also be used at this step to select RNA of certain lengths. For example, you can use a filter that will bind to RNA that is 18 nucleotides or longer. Everything that is <18 nucleotides will flow through the filter, and your RNA of interest will be kept on the filter. Lastly, the RNA is then mixed with an aqueous buffer, causing the RNA to re-dissolve in the solution.
While this process can be done manually, many laboratories choose to buy a kit that contains all of the reagents, which provides accuracy and consistency across the samples. It’s important to note that extreme care must be taken in sample handling. RNases are abundant enzymes in the environment which can degrade the RNA in the sample. It is important to use RNase-free solutions, glassware, and pipette tips. The use of phenol and chloroform also inhibits RNases from degrading your RNA.
The process of RNA extraction is possible through the basic chemical properties of RNA. By using the water-attracting properties of RNA, we can simply isolate it from cellular contents, precipitate it as a solid and then dissolve it back into solution. The final product, isolated RNA, can be used for a variety of downstream applications, such as quantifying the RNA and identifying RNA via sequencing.
