Knowledge Sharing | Basics of Nucleic Acid Extraction (Part 1)

From the perspective of nucleic acid extraction type, nucleic acid extraction experiments can be divided into DNA extraction , RNA extraction and DNA and RNA co-extraction Three types

DNA extraction

During the experiment, in order to obtain pure DNA, RNase is usually added during the extraction process to degrade the RNA in the sample and obtain relatively pure DNA. This method is widely used in various biological research and clinical diagnosis fields.

RNA extraction

Total RNA extraction and miRNA extraction are two different RNA extraction methods, which are mainly used for gene expression analysis, RNA sequencing, siRNA synthesis and functional research, RNA interaction research, molecular diagnosis, etc. Total RNA extraction includes rRNA, mRNA and small RNA such as tRNA, 5S rRNA, miRNA, etc., while miRNA extraction focuses on the separation and purification of miRNA, which is usually used to study its role in gene regulation.

Co-extraction of DNA and RNA refers to the process of isolating DNA and RNA from the same biological sample at the same time. This method is very important in molecular biology research as it can avoid bias caused by variations in lysis and extraction efficiency, especially in complex environmental samples. Co-extraction methods often require optimization of the extraction process to ensure the structural integrity and purity of the nucleic acids.

PART 2 Principles

1. Ensure the integrity of nucleic acid structure;

2. Eliminate contamination from other molecules;

3. Choose the appropriate extraction method;

4. Sample processing should be reasonable.

Cell lysis : There are various methods for cell lysis, including mechanical methods, chemical methods, enzymatic methods, etc.

Isolation and purification of nucleic acids : Separate nucleic acids from the mixture to obtain pure nucleic acids.

Concentration and precipitation of nucleic acids : Ethanol precipitation is the most commonly used method for nucleic acid concentration and precipitation.

PART 4 Extraction method

In nucleic acid extraction, different methods are usually required to process samples according to the type of samples in order to obtain nucleic acids with higher purity. For example, medical samples such as blood, animal tissues and cultured cells require specific preservation methods, while special medical samples such as serum, plasma and nasopharyngeal swabs require the use of special preservation fluids to inhibit enzyme degradation. In addition, plant samples are divided into common and polysaccharide polyphenols, and microbial samples are divided into bacteria and fungi, which need to be broken or lysed before extraction.

Common nucleic acid extraction sample types

01 Common animal samples ——Blood, animal tissue, cultured cells, etc.

This type of sample contains a larger number of cells and a simpler sample composition, but the processing method is also slightly different.

blood : The red blood cells without nuclei need to be separated or lysed before extraction.

Animal tissue: Grind after drying or grind directly with liquid nitrogen.

Cell samples ：Adherent cells need to be digested with trypsin and collected, and suspended cells need to be directly centrifuged and the supernatant discarded.

This type of sample has a small number of cells and relatively complex composition. During sampling and storage, a special preservation solution is usually required to inhibit the degradation of the sample by nucleases to ensure a high success rate of the experiment.

FFPE samples must first be dewaxed with xylene or mineral oil.

03 Plant samples ——Ordinary plant samples and polysaccharide and polyphenol plant samples

In addition to the cell wall structure, ordinary plants also have organelle structures such as vacuoles and chloroplasts inside, and their composition is relatively complex. Before extraction, they should be thoroughly ground with liquid nitrogen, and at the same time, the samples should be kept at low temperatures to avoid DNA degradation.

Polysaccharide and polyphenol plant samples such as fruit pulp and plant seeds have the same physical and chemical properties as nucleic acids, and polyphenols are easily bound to nucleic acids after oxidation, which affects the extraction of DNA. Usually, special reagents are needed to remove polysaccharides and polyphenols during the extraction process to obtain higher purity DNA.

04 Microbial samples ——Bacterial samples and fungal samples

Bacterial samples contain cell walls, and some also have flagella and capsules, which need to be broken down with lysozyme before extraction.

Fungal samples have cell walls mainly composed of polysaccharides and relatively complex internal cell components, and usually require enzymatic cell wall breaking, liquid nitrogen freeze-thaw, or ultrasonic lysis before nucleic acid extraction.

05 Environmental samples ——Soil, sewage, etc.

Depending on the nature of the environmental sample, it is necessary to remove relevant impurities before proceeding with the nucleic acid extraction step.