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Nucleic acids

High quality RNA from stable samples

The ability to analyze both RNA and protein from a single sample are required in some studies. This can be problematic since the two analytes have different requirements in order to be preserved in a high quality state. Standard RNA preservation strategies, e.g. RNA later, while preserving RNA quality do not prevent the more rapid changes to proteins and their modifications.

Heat stabilization is an efficient way to preserve proteins and their modifications but the issue of RNA quality have been a concern in such samples. Although heat inactivation does not stabilize RNA it does not affect the mRNA quality and enables a combination protocol where proteins and their modifications can be preserved by heat stabilization follwed by a second step where mRNA is preserved using standard RNA preservation strategies.

mRNA quality is not affected by heat treatment

mRNA quality assessed using QPCR, measure the difference in Ct between two QPCR reactions, a long and a short amplicon, from the same transcript. This is a more direct and meaningful measure compared to the traditional methods such as electrophoretic analysis of rRNA, e.g. Bioanalyzer assay, based on rRNA which only indirectly measures RNA quality. 

Schematic of principle for QPCR based quality measurement of RNA.

QPCR have been used to measure mRNA quality with three different QPCR based assays based on three different mRNAs reflecting different modes of degradation;

  • 18S assay for assessment of physical degradation.
  • Degradation assay for assessment of enzymatic degradation.
  • Stability assay for general mRNA stability.

All three QPCR based assays show comparable mRNA quality between heat stabilized samples and traditionally snap frozen samples, showing that mRNA is not affected by the short heating in the Stabilizor™ system.

∆Ct for three QPCR RNA quality assays in heat stabilized vs. snap frozen samples.

Application note, High quality RNA from heat stabilized samples