DNA AND RNA EXTRACTION – A COMPARISON

WHAT IS DNA EXTRACTION?

DNA extraction is the procedure of purification and isolation of DNA. There are three steps in DNA extraction. The steps are:

1. Cell lysis
2. Isolation
3. Precipitation

To find DNA, cell membrane barriers (nucleus membrane and cell membrane) break open in the process of cell lysis. Once done, membrane lipids are removed from the sample. Finally, the last step (precipitation) is reached, and here, proteins are removed from DNA by protease.

For DNA extraction, a DNA extraction kit consists of a nontoxic and straightforward method for isolating DNA from tissue, cultured cells, and whole blood. 

Following are the basic protocols of DNA extraction kit:

1. Cell lysis buffer to lysis cell membranes.
2. Broken down lipids with surfactants and detergents. 
3. Adding RNase for RNA digestion. 
4. Adding concentrated salt for RNA, lipids, digested proteins, and cell debris separation. 

To separate DNA from proteins, Phenol-chloroform extraction is a helpful method. This method is quick and straightforward. Here, alcohol (phenol) neutralizes proteins from the sample and leaves DNA as remains in the aqueous phase once the extraction ends. During the organic phase, denatured protein can be found.

Minicolumn purification is another method for DNA extraction. In this method, pH and salt concentration of buffer is essential for binding of DNA. 

WHAT IS RNA EXTRACTION?

Like DNA extraction, RNA extraction is the process of extracting RNA from given samples. The conventional RNA extraction kit or method is known as Guanidinium Thiocyanate-Phenol-Chloroform Extraction (1). In this method, Guanidinium Thiocyanate is used to denature the protein. It also disrupts hydrogen bonding in water and acts as a chaotropic agent. A unique and extra reagent is also present in different RNA extraction methods. This agent is called Tri-reagent. This single reagent contains phenol, sodium acetate, and guanidinium thiocyanate. 

 The basic structure protocol of RNA extraction is similar to that of DNA testing. The RNA extraction protocol is listed as follows:

1. To maintain the cell’s osmolarity, we wash cells with iced PBS. 
2. Cells and homogenize are aspirated with tri-agents. 
3. Mix some chloroform and shake. 
4. During shaking, three layers will form. The top layer is the water layer, and it is transparent. The middle layer contains DNA. The bottom layer is pink, and it’s organic. 
5. First, remove the top layer and add some isopropanol. When you shake it, a pellet will form. 
6. Now, wash that pellet with ethanol (75%) and then air dry the pellet. 
7. Now, use TE buffer or water to dissolve the pellet. 

RNA extraction commonly has a pH level below 7. RNA usually stays in the water phase acidic pH level. 

SIMILARITIES IN RNA AND DNA EXTRACTIONS:

1. Both RNA and DNA are nucleic acids. Therefore, their extraction processes generally consist of the purification and isolation of nucleic acids from samples. 
2. Both extractions involve cell lysis, isolation, purification of nucleic acids, and extract preparations. 
3. Cold conditions are required for both extractions. 
4. Both procedures involve centrifugation during separation in the mixture. 
5. The method of phenol-chloroform extraction is used in both procedures. 
6. Guanidinium Thiocyanate is suitable for both extraction procedures to protect nucleic acids. 

BASIC DIFFERENCES BETWEEN DNA AND RNA EXTRACTIONS:

As the name suggests, RNA extraction is the extraction, purification, and isolation of RNA from samples. On the other hand, DNA extraction is the process of purification and isolation of DNA. 

The pH level is one of the main differences between DNA and RNA. In DNA extraction, the procedure ends at pH 8, while in RNA procedure, the extraction ends at pH 4.7.

The steps of each procedure have slight differences. For example, both DNA and RNA start with cell lysis, but DNA extraction’s next step is to remove membrane lipids, and then DNA precipitating. On the other hand, RNA extraction goes from cell lysis to guanidinium extraction and preparation with isopropanol. 

CONCLUSION:

DNA quickly denatures acidic pH at the organic phase. The procedure is carried out at pH 8. However, the pH level of RNA extraction is lower to prevent degradation. The basic structure and purpose of both extraction procedures are similar (purification of nucleic acid), and the main difference is pH levels. 

Bibliography

1. The single-step method of RNA isolation by acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction: Twenty-something years on. Sacchi, Piotr Chomczynski & Nicoletta. 2006.