Abstract
Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.
Highlights
The removal of a biological sample from its in vivo environment initiates a series of signaling cascades as the cells in the sample adapt to their new environment [14]
The RNA yield, purity and quality has been measured in RNA extracts from heat stabilized and snap frozen brain tissue samples with the aim of investigating the effect of heat denaturation on two different RNA quality measurements, either Bioanalyzer or qPCR
Analysis of multiple analytes from precious biological samples can be challenging as different analytes require different preservation measures
Summary
The removal of a biological sample from its in vivo environment initiates a series of signaling cascades as the cells in the sample adapt to their new environment [14]. E.g., peptides, protein phosphorylation, lipids and metabolites, will start to change from their actual in vivo levels, distorting analysis results and making interpretation of analytical results difficult or even impossible [5,15]. A range of preservation techniques are used to deal with post-sampling change but they are usually optimized to preserve a specific analyte or class of analytes while not preserving other analytes. This approach is generally satisfactory as long as only analytes from a single analyte class are analysed. Karlsson et al / Biomolecular Detection and Quantification 7 (2016) 21–26
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