Tips and Tricks for RNA Isolation

RNA isolation can be a demanding procedure in molecular biology. Researchers often face RNA degradation, low yield or purity, and unwanted DNA contamination. Different biological materials present their own challenges. For example, microbes such as gram-positive or gram-negative bacteria, fungi, and archaea may have robust cell walls that are difficult to break. Samples like faeces or plants can contain inhibitory compounds such as polyphenolics, humic or fulvic acids, and tannins that may co-precipitate with RNA and compromise downstream applications including RT-qPCR.

In this guide, experienced researchers share practical advice for improving RNA isolation results and recovering high-quality, DNA-free RNA.

RNA molecules are inherently unstable and degrade quickly when exposed to RNases. Many sample types naturally contain high levels of these enzymes. The most effective way to preserve RNA integrity is to stabilise the material as soon as it is collected.

Common preservation approaches include snap freezing in liquid nitrogen, immersion in a dry-ice ethanol bath, or storage at –80 °C. While effective, these methods can cause freeze–thaw damage to nucleic acids and may not be practical during fieldwork or at remote collection sites.

Recommended RNA preservation strategies:

• Dissolving the sample directly into a lysis buffer that inactivates RNases, such as TRIzol® or RNA Lysis Buffer. The material can then be processed immediately or stored at low temperature.

• Placing the sample in a stabilisation reagent such as DNA/RNA Shield, which protects RNA at room temperature for extended periods. This is particularly useful when handling valuable clinical material such as biopsies or whole blood, or when working outside a laboratory environment.

Efficient cell disruption is essential for high RNA yield and quality. However, not all cell types respond equally to the same method. Blood-derived cells such as lymphocytes or PBMCs and microbial cells often require more than a detergent-based buffer.

Better results can be achieved by combining chemical lysis with mechanical disruption such as bead beating, or by including an enzymatic treatment such as proteinase K or lysozyme before extraction.

Complete lysis also supports smoother processing in column-based RNA isolation methods. Without it, column clogging, buffer carryover, incomplete DNA removal, and poor elution can occur. Zymo Research has tested and optimised lysis protocols suitable for a wide variety of sample sources.

A frequent challenge in RNA isolation is residual DNA. This contamination can affect absorbance-based quantification such as Nanodrop readings, giving artificially high RNA concentrations. It can also interfere with sensitive downstream analyses such as RNA sequencing.

DNA can be removed through TRIzol® phase separation, by using DNA removal columns, or by applying DNase treatment during the extraction process.

Visual inspection of RNA on an agarose gel or with an Agilent TapeStation® can quickly reveal high-molecular-weight DNA above the 28S ribosomal RNA band (Figure 2). For applications with high sensitivity to DNA, qPCR or Qubit assays are recommended.

Many Zymo RNA isolation kits integrate on-column DNase I treatment, which saves time by eliminating the need for separate clean-up steps after extraction. This approach helps ensure that the RNA is ready for immediate use in downstream applications.

No single method works for all types of RNA extraction. The choice of kit depends on the sample material, input amount, and desired throughput. Zymo Research provides a range of kits adapted to different needs, from small-scale micropreps to high-throughput magnetic bead formats.

The table below lists recommended products for various sample types, which may help when comparing types of RNA extraction methods.