An Overview of RNA Isolation Kits

RNA isolation is an important initial step in experiments for gene expression and regulation, such as quantitative PCR, RNA-seq, and in-situ hybridization. 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 RNA purification kits are available today that can improve efficiency, reproducibility, and yield in collecting various types of RNA.

What are examples of RNA types that can be purified?

• Total RNA - As its name implies, total RNA includes all the RNA subtypes extractable from a given cell or tissue sample. This includes mRNA, rRNA, tRNA, small regulatory RNA, and others.
• mRNA - Messenger RNA (mRNA) contain the protein-coding information of genes. They are the desired starting material for experiments in gene expression analysis, such as mRNA-seq and qPCR.
• miRNA - MicroRNA (miRNA) are a type of small, noncoding RNA that play a large role in gene regulation, particularly gene silencing. Circulating miRNA in the blood are also being studied as disease biomarkers.
• Viral RNA - RNA released by viruses can be purified in order to identify and characterize the infectious agent. These viral RNA particles can be found circulating in blood, saliva, bronchoalveolar lavage, and other bodily fluids.

How do RNA isolation kits work?

RNA isolation and purification kits contain a complete set of buffers, reagents, and plasticware for the rapid and efficient collection of RNA. These sets include chemical lysis buffers, RNA-binding beads or resins, and columns. They are intended to be optimized, RNase-free, and can include accessory reagents, such as DNases, RNase inhibitors, or RNA stabilizers, to ensure pure and robust RNA yields even from small amounts of starting material. Many kits also use methods that omit phenol, which is a hazardous volatile solvent.

Column-based purification kits use immobilized silica to bind RNA, which can be eluted using centrifugation. Magnetic bead-based kits use a magnetic field anchor RNA so impurities can be removed in the supernatant. The purification method will further vary depending on the RNA type or RNA source. For example, mRNA isolation kits generally employ labeled or bound oligo(dT) fragments to target the poly(A) tails of RNA. RNA isolation kits for fixed tissues will also include additional reagents to account for any crosslinks or modifications introduced during fixation.

When choosing a kit, consider the following factors unique to your needs: the type and source of RNA, the amount of starting material, the amount of desired RNA yield, the throughput or number of samples needed to be purified, and whether to use a centrifuge- or magnet-based method.