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 degrees 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 degrees 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 degrees C could preferentially be used. This may open opportunities for monitoring gene expression changes in clinical settings.
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 degrees 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 degrees 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 degrees C could preferentially be used. This may open opportunities for monitoring gene expression changes in clinical settings.
Authors: Anastasios Mastrokolias; Johan T den Dunnen; Gertjan B van Ommen; Peter A C 't Hoen; Willeke M C van Roon-Mom Journal: BMC Genomics Date: 2012-01-18 Impact factor: 3.969
Authors: Kristina Vartanian; Rachel Slottke; Timothy Johnstone; Amanda Casale; Stephen R Planck; Dongseok Choi; Justine R Smith; James T Rosenbaum; Christina A Harrington Journal: BMC Genomics Date: 2009-01-05 Impact factor: 3.969