RNA isolation is also called: RNA extraction, RNA purification or Trizol extraction

​RNA Isolation: what is it and how does it help my studies?

RNA isolation is the process by which RNA is separated from viruses, cells, or sample matrices. RNA (ribonucleic acid) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA is often extracted for the purpose of downstream PCR applications.

​RNA extraction differs from isolation of other nucleic acids because of the ubiquitous presence of RNases, that cleave RNA and decrease its diagnostic utility. These enzymes can renature even post-autoclaving, regaining activity and degrading the RNA of interest. There are several RNase inhibitors available in the market such as diethyl pyrocarbonate (DEPC) to counteract these enzymes.
​There are several types of RNA in prokaryotes and eukaryotes. For RNA purification, it is important to note the abundance of each type. Ribosomal RNA (rRNA) being 80-90%, messenger RNA (mRNA) being 2.5-5% and variable amounts of Transfer RNA and small nuclear RNAs. Isolation of RNA can be done with different chemistries, each resulting in different RNA profiles. Below are three scientific manuscripts that highlight how traditional RNA isolation / RNA extraction / RNA purification approaches bias the resulting RNA profile.
  • Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal
    • Rikki A. M. Brown, Michael R. Epis, Jessica L. Horsham, Tasnuva D. Kabir, Kirsty L. Richardson & Peter J. Leedman BMC Biotechnology volume 18, Article number: 16 (2018)  - Link to manuscript
  • A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma​
    • Ryan K.Y. Wong, Meabh MacMahon, Jayne V. Woodside & David A. Simpson BMC Genomics volume 20, Article number: 446 (2019)  - Link to manuscript
  • RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition 
    • Ana McCarthy, Edna Chiang, Marian L. Schmidt, Vincent J. Denef, PLOS ONE (2015)  - Link to manuscript

How is RNA isolated?

  1. Trizol / Organic RNA isolation uses detergent or phenol in the presence of high salt or RNase inhibitors. Once the cells are lysed, proteins can be extracted using mixture of phenol : chloroform : isoamyl alcohol (25:24:1) solution effectively extracts RNA. Chloroform enhances the extraction of the nucleic acid by denaturing proteins and promoting phase separation. TRIzol offers a all-in-one reagent for lysing and separating proteins and nucleic acids from a sample. After phase separation, the upper aqueous phase containing the RNA is removed to a clean tube, and the RNA is recovered by using ethanol or isopropanol precipitation.  
  2. Solid-phase RNA isolation uses beads or columns to capture and release RNA at desired times. The initial procedure is similar to organic isolation, where a lysate is first obtained but the denaturing conditions of the buffer must be lowered before the lysate is applied to the extraction column. This sample is incubated with binding beads or columns, that selectively bind to RNA and thereby allows for the removal of proteins, salts, PCR inhibitors, and other contaminants. Magnetic beads or columns are often used in automated systems and are available in convenient RNA isolation kits.
    1. Magnetic beads selectively bind to RNA in the lysate and are then separated from the lysate using a magnet. These beads are washed to remove contaminants, before the RNA is  eluted off of the beads, resulting in isolated RNA. 
    2. Columns selectively bind to RNA in the lysate and allow non-RNA components of the lysate to flow through the column. The column is then washed to remove contaminants, before the RNA is  eluted off of the column, resulting in isolated RNA. 

Solid-phase RNA isolation has multiple advantages over Trizol / organic RNA isolation methods. For instance, the isolation procedure becomes easier and gentler (no phenol used), beads can be conjugated to enzymes, antibodies, and even custom ligands. Additionally automation with magnetic beads confers great scalability, high throughput and decreased contamination with RNases. 
Direct PCR / extraction-free PCR and the improved, Next Generation Direct PCR, allows for downstream PCR applications without the need for RNA extraction / RNA purification. Furthermore, direct PCR does not alter the RNA profile of your sample since it does not use 'capture and release' technology or separate components of your sample with organic solvents like Trizol, as used by solid-phase and organic RNA isolation.​

Stay up to date on new developments

Check out our blog and press page.

Related topics that may be of interest:

​Need help with your project?
Let our R&D team help you
[email protected]