Abstract
Background: Based on quantification of relative changes in lipopolysaccharide (LPS)-regulated mRNA transcripts, the present study aimed to establish a robotic method to isolate RNA from stabilized frozen whole blood suitable for gene expression analysis.
Methods: Whole blood (±LPS) was stored in EasyLyse™ solution or PAXgene® tubes (room temperature and −70°C) for comparison of storage methods, then subjected to robotic isolation of total RNA. Yield, quality and relative changes in 11 selected mRNA transcripts were examined. Method precision (% coefficient of variation) for a longitudinal control was established. The influence of globin mRNA from reticulocytes in quantitative RT-PCR was examined.
Results: All storage methods gave a similar high-quality RNA yield. The differences in the 11 specific mRNA quantities stored in EasyLyse or PAXgene® at −70°C were small: mean −0.01 (95% CI –0.19 to 0.17). The CV for mRNAs in the longitudinal control ranged from 3% to 150%. Thus, the number of replicates necessary to detect a 20% difference (power 0.8) ranged from 2–50. Globin mRNA had no influence on quantitative RT-PCR
Conclusions: Based on measuring the relative changes in specific mRNAs in LPS-exposed whole blood, we conclude that PAXgene® tubes stored at −70°C could preferentially be used. This may open opportunities for monitoring gene expression changes in clinical settings.
Clin Chem Lab Med 2007;45:171–6.
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