Quantification of relative changes in specific mRNAs from frozen whole blood - methodological considerations and clinical implications.
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.
Øvstebø
Reidun
R
R&D Group, Department of Clinical Chemistry, Ullevål University Hospital, Oslo, Norway. reidun.ovstebo@medisin.uio.no
Lande
Knut
K
Kierulf
Peter
P
Haug
Kari Bente Foss
KB
D001769:Blood; D001793:Blood Preservation / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D005914:Globins / Q000235:genetics; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008722:Methods; D012333:RNA, Messenger / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D012156:Reticulocytes; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012371:Robotics / Q000379:methods
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D005914:Globins