Sample to Insight

Look inside the red box — get the RNA yield and integrity you need

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Overcoming the five main issues in RNA preparation

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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)