Improving RNA yield and quality is easy with the right protocols. This trouble shooting guide presents methods to overcome common difficulties in RNA purification. Prevent RNA degradation, improve RNA yields, reduce genomic DNA contamination, and more.

1. Sample to Insight
Look inside the red box — get the RNA yield and integrity you need
Find your stabilization and purification solution for every sample type at www.qiagen.com/RNA
Overcoming the
five main issues in
RNA preparation
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, QIAzol®
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Challenge Reason What to be done?
RNA Degradation • RNases still active in the sample
• Contamination with RNases during sample preparation and RNA purification
• Work in an RNase-free space
• Use RNase-free buffers and tips only
• Immediately transfer sample to liquid nitrogen and store at low temperatures
• Stabilize at room temperature with appropriate stabilization reagent
Low RNA yield • Insufficient disruption and homogenization of sample
• Poor sample quality
• Suboptimal purification technology
• Overloaded columns
• Low RNA content in the sample (e.g., primary cells versus cell lines)
• Pellet lost during precipitation step
• Choose appropriate disruption and homogenization method
• Choose optimal RNA purification kit and protocol
• If possible, improve sample quality
• Avoid homebrew methods that include a precipitation step
gDNA contamination • Suboptimal gDNA removal (especially with phenol-based methods)
• Overloading columns
• Choose appropriate gDNA removal method, for phenol-based techniques choose QIAzol®
with gDNA
Eliminator Solution
• Follow recommended protocol steps (correct temperature QIAzol phase separation; no overloading of columns)
Low OD ratios • Phenol contamination leading to overestimation of RNA concentration
• Sample rich in secondary metabolites
• Always use phenol-based approaches (e.g.,QIAzol) with RNeasy®
• Always choose optimal purification methods (RNeasy) and follow recommended protocol (no overloading;
sufficient drying of membranes from ethanol)
• Check guidelines for OD interpretation. See what can and cannot be concluded from them
Bad results due to unsuitable protocol or technology • Chosen technology gives suboptimal performance with specific sample material • Use specific RNeasy kits and protocols for your sample type (cell type, whole range of tissue types, and plant,
bacteria, yeast, and FFPE samples)