RNA plays a vital role in many biological processes. Preparing isolated and purified RNA from biological samples such as cells and tissues is thus a critical step in many scientific experiments, including gene expression/regulation analysis, RNA sequencing, and PCR. In this article, we will discuss the importance of RNA extraction and some of the most widely utilized methods currently available for RNA isolation and purification.
The Importance of RNA Extraction
Isolating and purifying RNA is essential for accurate and reliable analysis of gene expression and other RNA-based processes. RNA extracted from biological samples should be free of contaminants (e.g., DNA and proteins) and should have high quality and integrity. Contaminants can interfere with downstream applications like PCR and RNA sequencing, leading to inaccurate results. High-quality RNA samples enable researchers to accurately analyze gene expression levels, identify differentially expressed genes, and study RNA structure and function.
RNA Extraction Methods
Trizol There are several methods available for extracting RNA from biological samples. Among the more popular is guanidinium thiocyanate-phenol-chloroform extraction, also known as the Trizol method. Trizol is a monophasic solution of guanidinium thiocyanate and phenol, which effectively denatures proteins and inactivates RNases. Addition of chloroform creates a biphasic system in which DNA and protein partition into the lower organic phase while RNA is retained in the upper aqueous phase. The RNA is subsequently precipitated from the aqueous phase using isopropanol.
Spin columns Silica membrane-based spin column extraction is another common RNA extraction method. The silica membrane selectively binds to RNA and allows other cellular components to pass through. After binding to the membrane, RNA is washed and eluted from the column with a high-salt buffer.
Magnetic beads Magnetic bead-based extraction is a relatively new method that has gained traction in recent years. This method employs magnetic beads that are coated with oligo-dT, which selectively binds to poly(A) tails of mRNA molecules. The beads are then separated by means of a magnetic field, and the RNA is eluted from the beads.
A More Affordable Approach
Despite their popularity, these methods have several drawbacks. The Trizol method can be time-consuming, as it involves several steps, including homogenization, chloroform extraction, and isopropanol precipitation. Additionally, the use of hazardous chemicals such as phenol and chloroform can pose health risks to laboratory personnel. Oligo-dT-coated beads are highly specific for mRNA, and may not be appropriate for other RNA molecules.
The EpiQuik™ Total RNA Isolation Fast Kit(catalog #P-9105) and the EpiQuik™ Magbeads Quick RNA Isolation Kit(catalog #P-9106) provide a fast, simple, and cost-effective alternative for isolating total RNA from mammalian cells and tissues, in just 30 minutes. These kits are complete sets of optimized buffers and reagents for the entire extraction procedure, from cell lysis to RNA purification, making them the convenient and economical option. Detergents are used to lyse cells and inactivate RNase. The specialized high-salt buffering system allows for binding of RNA to the silica magbeads (P-9106) or the glass fiber matrix of the spin column (P-9105) and for removal of protein/DNA. Contaminants and impurities are efficiently washed away, and pure RNA is eluted. The RNA extracted with these kits is suitable for a variety of routine applications: RT-PCR, cDNA synthesis, northern blotting, differential display, primer extension, and mRNA selection.
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