Overview of RNA Isolation

October 25, 2024

RNA isolation is an important initial step in the experimental analyses of gene expression and regulation in methods such as quantitative PCR, in-situ hybridization, and RNA-Seq. However, the extraction and purification of RNA can pose a challenge due to its delicate nature. This is partly owed to the extra hydroxyl group on its pentose ring, which confers greater reactivity and a higher susceptibility to hydrolysis. Ribonucleases are also prevalent in cells and tissues that, if not addressed, can quickly degrade RNA. A wide variety of commercial RNA purification kits are available today that can improve efficiency, reproducibility, and yield in collecting various types of RNA. In this overview guide and catalog, we highlight the various types of RNA isolation kits and offer useful tips in product selection.

How do RNA isolation kits work?

RNA isolation kits contain complete sets of buffers, reagents, and other consumables for the rapid and efficient collection of RNA. While isolation procedures and components will vary depending on the intended RNA type or sample type, a general workflow may proceed as follows. To begin, a lysis buffer containing detergents and enzymes is added to the sample to break down cell membranes and release RNA. Often, a DNA removal step is performed using differential binding conditions and/or digestion with DNase.

RNA in the lysate will bind to an RNA-binding surface (such as silica) that is utilized in column or magnetic formats. In column-based purification kits, silica is immobilized in a membrane or resin, allowing solutions to pass using a centrifuge or vacuum. Meanwhile, magnetic bead-based kits employ a magnetic separator to anchor RNA, allowing impurities to be removed in the supernatant. In either method, the purified RNA is ultimately collected in a final elution step.

RNA purification kits use standardized and optimized reagents free of RNases. They also often include accessory reagents, such as RNase inhibitors or RNA stabilizers, to ensure pure and robust RNA yields even from small amounts of starting material. Many kits use methods that omit phenol, which is a hazardous volatile solvent.

Common types of RNA purification kits

RNA purification kits can be classified according to the target RNA type or a particular sample type from which the RNA is extracted from. These are highlighted below:

Total RNA - Total RNA encompasses the full range of RNA subtypes found within a cell or tissue sample, including mRNA, rRNA, tRNA, miRNA, and other small RNA. These kits are usually designed to extract RNA from specific sample types (such as blood, tissue, plant) and can be further categorized. Note that ribosomal RNA (rRNA) is the most abundant component of total RNA and additional processing will be required when studying specific RNA, such as transcripts.
mRNA - High-quality isolation of messenger RNA (mRNA) is important in downstream analyses of gene expression, such as mRNA-seq and qPCR. mRNA purification kits often use oligo(dT) hybridization methods for specific binding of poly-A tails, a defining characteristic of mature mRNA (with exceptions in bacteria). Some protocols may also include additional steps to remove non-target RNA.
miRNA - MicroRNA (miRNA) are a type of small, noncoding RNA that play a large role in gene regulation, particularly gene silencing. The study of circulating miRNA as disease biomarkers is also an active area of research. While more generalized RNA kits are optimized for isolating larger RNA molecules, miRNA isolation kits are better suited for enriching RNA under 200 bases.
Blood RNA - Blood, including serum and plasma, is a complex matrix containing cells, proteins, anticoagulants, RNases, and other components. Blood RNA isolation kits are designed to effectively lyse blood cells and remove contaminants. They can also be useful for isolating RNA from blood-borne pathogens.
FFPE RNA - Formalin-fixed, paraffin-embedded (FFPE) tissue, while useful for sample preservation, are challenging sources of RNA for extraction. FFPE RNA isolation kits include specific protocols and reagents optimized for the unique challenges of FFPE samples, such as the need to reverse cross-linking and remove paraffin.
Viral RNA - Viral RNA particles can be found circulating in blood, saliva, bronchoalveolar lavage, and other bodily fluids. Viral RNA extraction kits generally work by first enriching for viral particles, followed by RNA isolation.
Plant RNA - Plant tissues offer unique difficulties in RNA extraction, such as tough cell walls and the significant presence of starch, proteins, and fiber. Plant RNA extraction kits are optimized to overcome these tissue-specific challenges.

PolyATtract mRNA Isolation System I from Promega

mRNA Isolation Kits

Viral RNA Isolation Kit from ScienCell Research Laboratories

Viral RNA Purification

FastPure Cell/Tissue Total RNA Isolation Kit V2 RC112 from Vazyme Biotech

Cell/Tissue RNA Purification

Aurum™ Total RNA Fatty and Fibrous Tissue Kit from Bio-Rad

Total RNA Purification

RNA isolation kit considerations

One main takeaway from this guide can be summed up as selecting the appropriate extraction kit for the specific RNA or sample type. However, there are some important factors to consider when choosing the right kit.

Plan ahead for the downstream application. Ultimately, the success of an RNA extraction is not merely determined by the yield or purity of the collected RNA, but by the feasibility of this material for use in downstream experiments. Purified RNA with significant amounts of rRNA, for example, will not be optimal for RNA-seq and transcriptomic analyses. Contaminants (such as RNases, DNA, plant-derived phenolic compounds) that linger in purification can degrade RNA or interfere with downstream assays. The ideal kit will address these issues, leading to the most optimized outcome for experimentation. In addition to using the appropriate kit for the right sample type, knowing the right target conditions for the RNA product can help in the deeper selection of the best fit kit.

Consider the format. As mentioned above, RNA extraction kits commonly utilize column or magnetic bead formats. Spin columns, which generally only require a benchtop centrifuge, typically use simple, quick protocols that yield good, high-quality RNA from small sample sizes. Their drawback comes from low throughput and some susceptibility to overloaded filters from too much sample or incomplete lysis. Larger chromatography columns can be used to accommodate more starting material and produce greater yields. Magnetic beads which can be distributed in multi-well plates (eg. 96-wells) are great for increasing throughput and implementing automation-friendly workflows. This format may be the ideal choice when screening or dealing with many samples.

Do the due diligence and review the protocol. Reviewing the protocols or product manuals when available can be valuable when selecting an RNA purification kit. These documents provide essential details, such as the complexity of the steps, total processing time, and any special equipment needed (e.g., centrifuges, vacuum manifolds, or magnetic stands). They also help determine whether the kit is compatible with your sample type, what RNA yield to expect, and if further processing is necessary for downstream applications. An ideal protocol should strike a balance between efficiency and ease of use, saving time while ensuring high-quality RNA extraction that fits the needs of your specific experiment.