The main difference between DNA and RNA extraction is that the pH level of DNA extraction is pH 8 whereas the pH level of RNA extraction is pH 4.7. DNA tends to denature and move to the organic phase at acidic pH. At alkaline pH, the RNA undergoes alkaline hydrolysis due to the presence of 2′ OH in the ribose sugar.
DNA and RNA extraction are the two procedures involved in the isolation and purification of nucleic acids from the cells of tissues. Both procedures consist of three steps. Good quality DNA and RNA play a critical role in molecular biology, biotechnology, genomics, and epidemiology experiments.
Key Areas Covered
1. What is DNA Extraction
– Definition, Procedure
2. What is RNA Extraction
– Definition, Procedure
3. What are the Similarities Between DNA and RNA Extraction
– Outline of Common Features
4. What is the Difference Between DNA and RNA Extraction
– Comparison of Key Differences
Key Terms: Cell Lysis, Denaturation of Proteins, DNA Extraction, pH, Precipitation, RNA Extraction
What is DNA Extraction
DNA extraction is the isolation and purification procedure of DNA. DNA can be isolated from blood, frozen tissue samples or paraffin tissue blocks. The three steps of DNA extraction are cell lysis, isolation of DNA, and precipitation. During cell lysis, cell membrane barriers such as cell membrane and the membranes of the nucleus break open in order to expose DNA. The next step is the removal of membrane lipids from the sample. Finally, the precipitation of DNA involves the removal of DNA-associated proteins by protease and the removal of RNA by RNase.
Basic Protocols of DNA Extraction
Shown below are the basic protocols of DNA extraction.
1. Cell lysis with the cell lysis buffer to lyse cell membranes
2. Lipids are broken down with detergents and surfactants
3. Digestion of proteins by adding protease
4. Digestion of RNA by adding RNase
5. Separation of cell debris, digested proteins, lipids, and RNA by adding concentrated salt followed by centrifugation
6. Ethanol precipitation of DNA with ice-cold ethanol or isopropanol. The ionic strength of sodium acetate can be used to improve precipitation. The precipitated DNA appears as threads in the final solution.
Phenol-chloroform extraction can also be used to separate DNA from proteins. Here, phenol denatures the proteins in the sample, and the DNA remains in the aqueous phase at the end of the extraction. And, in in the organic phase, you can find the denatured proteins. Another method to extract DNA is the minicolumn purification. Here, binding of DNA into the column relies on the pH and the salt concentration of the buffer.
What is RNA Extraction
RNA extraction refers to the process of purifying RNA from samples. The conventional method of RNA extraction is called the guanidinium thiocyanate-phenol-chloroform extraction. Guanidinium thiocyanate denatures proteins. Moreover, it disrupts hydrogen bonding of water molecules and serves as the chaotropic agent. A special reagent is used in the RNA extraction called the Tri-reagent. It contains guanidinium thiocyanate, phenol, and sodium acetate. The purpose of the basic steps of RNA extraction is similar to that of in DNA extraction. The protocol for RNA extraction is described below.
1. Cells are washed with ice-cold PBS to maintain the osmolarity of cells.
2. Aspirate the cells and homogenize the sample with Tri-reagent.
3. Add chloroform and shake.
4. Centrifugation may result in three layers. The top layer is the aqueous layer, which is clear. The middle layer or the interphase contains the precipitated DNA. The bottom layer is the organic layer, which is pink.
5. Remove the aqueous layer and add isopropanol. Centrifugation may result in a pellet.
6. Wash the pellet with 75% ethanol. Air dry the pellet.
7. Dissolve the pellet with TE buffer or water.
RNA extraction is generally carried out at pH below 7. At alkaline pH, RNA is more prone to be degraded by alkaline hydrolysis due to the presence of an OH group at 2′ position of the ribose sugar. In addition, RNA tends to remain in the aqueous phase at acidic pH. On the other hand, DNA tends to denature and move into the organic phase at acidic pH. Therefore, DNA extraction can be carried out at about pH 8. DNA is made up of deoxyribose sugar and does not undergo alkaline hydrolysis.
Similarities Between DNA and RNA Extraction
- DNA and RNA extraction are the procedures involved in the isolation and purification of nucleic acids from biological samples.
- Both procedures involve cell lysis, purification of nucleic acids from the debris and associated proteins, and preparation of the extracts.
- For both procedures, have to maintain cold conditions throughout.
- Involves centrifugation in the separation of components in the mixture.
- Need to inactivate the activity of nuclease enzymes during both procedures.
- Phenol-chloroform extraction is one of the critical steps of both types of extractions.
- Guanidinium thiocyanate can be used to protect nucleic acids.
- Precipitation of RNA can be done with isopropanol.
- The ionic strength of the sodium acetate is used to improve the precipitation of nucleic acids.
- The samples can be quantified by measuring the OD at 260 nm.
Difference Between DNA and RNA Extraction
DNA Extraction: Isolation and purification procedure of DNA
RNA Extraction: Process of purifying RNA from samples
Type of Nucleic Acid Extracted
DNA Extraction: DNA
RNA Extraction: RNA
DNA Extraction: Done at 8
RNA Extraction: Done at 4.7
DNA Extraction: Breaking the cell membranes or cell lysis, removing the membrane lipids, and precipitating the DNA
RNA Extraction: Cell lysis, guanidinium thiocyanate-phenol-chloroform extraction, preparation with isopropanol
Use of DEPC-Treated Water
DNA Extraction: None
RNA Extraction: All reagents are prepared with DEPC-treated water
DNA Extraction: DNA can be extracted prior and is stored in batches
RNA Extraction: RNA extraction is done just before the downstream procedures
DNA Extraction: At -20 °C
RNA Extraction: At -80 °C
DNA extraction is carried out at pH 8. DNA tends to move to the organic phase as it denatures at acidic pH. But, RNA extraction is carried out at low pH to prevent degradation by alkaline hydrolysis. The basic purpose of the steps of both DNA and RNA extraction is similar. The main difference between DNA and RNA extraction is the pH conditions used in each type of extraction.
1. “The Basics of DNA Extraction.” Alaska BioPREP Virtual Textbook, Alaska BioPREP University of Alaska Fairbanks, Available Here
2. “RNA Isolation and Reverse Transcription Protocol: Cells in Culture.” abcam, Available Here
1. “Figure 17 01 02” By CNX OpenStax – (CC BY 4.0) via Commons Wikimedia
2. “DNA Extraction” By Joo Nath – Own work (CC BY-SA 4.0) via Commons Wikimedia
3. “PhOH-CHCl3 extraction” By Squidonius (talk) – Own work (Original text: self-made) (Public Domain) via Commons Wikimedia