How to Ensure Max Yield & Purity in RNA Extraction

Extraction of high-quality and intact RNA from biological samples is an essential prerequisite for many downstream applications, including northern blotting, reverse transcription real-time PCR, cDNA synthesis, microarrays, and NGS transcriptome analysis. However, the extraction procedure can be complicated by the nature of the RNA molecule itself, such as its higher chemical instability and susceptibility to enzymatic degradation compared with DNA. Several factors should be taken into consideration to ensure maximal yield and purity of extracted RNA.

Inhibiting RNases

RNA can be rapidly degraded by the abundant ribonucleases (RNases) in cells and tissues. The extreme robustness of certain RNases makes their inactivation more challenging than DNase neutralization. Additionally, RNases are secreted by our skin, and several RNases are present in the surrounding environment. Proper sample handling and aseptic technique are therefore of paramount importance when isolating RNA.

Research tools like micropipettes should be designated solely for RNA extraction, thoroughly cleaned with chemical agents that destroy RNases, and maintained separately from other laboratory equipment. RNase-free solutions, pipet tips, and glassware should be used during the extraction. Special care must be taken to avoid contact with bare skin, which harbors RNases. RNase inhibitors (guanidine salts, SDS, phenol-containing compounds, or other strong denaturants) may be incorporated into extraction protocols to preserve RNA integrity.

Removing DNA and Ethanol Contamination

Residual DNA can affect the purity of RNA extracts. RNA extraction can be followed by DNase I treatment to remove contaminating DNA.

The presence of ethanol in the purified RNA can inhibit downstream enzymatic reactions. Ethanol should be completely removed after RNA wash steps.

RNA Extraction Methods

There are various methods for isolating RNA that employ different techniques, such as organic extraction, spin columns, and magnetic bead technology. The most popular of these is acid guanidinium thiocyanate-phenol-chloroform extraction, in which RNA is recovered from the upper aqueous phase of a multiphasic system while DNA, protein, and lipid partition into the lower organic phase. The addition of the chaotropic agent guanidinium thiocyanate to the organic phase aids in denaturing RNases and inhibiting RNA degradation. RNA is subsequently recovered from the aqueous phase by alcohol precipitation.

Column- and magnetic bead-based systems provide a more rapid and convenient alternative to organic extraction methods. EpiGentek offers a variety of magnetic bead and spin column kits for isolating RNA from diverse sample sources (cells, tissues, whole blood, saliva, nasopharyngeal swabs) and species (viral, mammalian, bacterial, fungal, plant). Detergents and chaotropic salt are used to lyse cells and inactivate RNases. A specialized high salt buffering system allows RNA bases to bind to the magnetic beads or to the glass fiber matrix of the spin column while contaminants pass through. Impurities are efficiently washed away, and the pure RNA is eluted with an aqueous buffer, without the need for phenol extraction or alcohol precipitation.

In light of the current events surrounding the COVID-19 pandemic and the associated SARS-CoV-2 virus, procedures for the quick preparation of viral RNA are of particular interest. EpiGentek recently developed the new EpiQuik Viral RNA Isolation Fast Kit and EpiMag Viral RNA Isolation Kit to deliver high-quality total viral RNA via spin columns and magnetic beads, respectively, in just 10-25 minutes from cell-free liquid specimens, specifically saliva and nasal or nasopharyngeal swabs.