Limited dynamic range of immune response gene expression observed in healthy blood donors using RT-PCR.

The use of quantitative gene expression analysis for the diagnosis, prognosis, and monitoring of disease requires the ability to distinguish pathophysiological changes from natural variations. To characterize these variations in apparently healthy subjects, quantitative real-time PCR was used to measure various immune response genes in whole blood collected from blood bank donors. In a single-time-point study of 131 donors, of 48 target genes, 43 were consistently expressed and 34 followed approximately log-normal distribution. Most transcripts showed a limited dynamic range of expression across subjects. Specifically, 36 genes had standard deviations (SDs) of 0.44 to 0.79 cycle threshold (C(T)) units, corresponding to less than a 3-fold variation in expression. Separately, a longitudinal study of 8 healthy individuals demonstrated a total dynamic range (> 2 standard error units) of 2- to 4-fold in most genes. In contrast, a study of whole blood gene expression in 6 volunteers injected with LPS showed 15 genes changing in expression 10- to 90-fold within 2 to 5 h and returning to within normal range within 21 hours. This work demonstrates that (1) the dynamic range of expression of many immune response genes is limited among healthy subjects; (2) expression levels for most genes analyzed are approximately log-normally distributed; and (3) individuals exposed to an infusion of bacterial endotoxin (lipopolysaccharide), show gene expression profiles that can be readily distinguished from those of a healthy population. These results suggest that normal reference ranges can be established for gene expression assays, providing critical standards for the diagnosis and management of disease.

Stabilization of mRNA expression in whole blood samples.

BACKGROUND: Accurate quantification of mRNA in whole blood is made difficult by the simultaneous degradation of gene transcripts and unintended gene induction caused by sample handling or uncontrolled activation of coagulation. This study was designed to compare a new blood collection tube (PAXgene Blood RNA System) and a companion sample preparation reagent set with a traditional sample collection and preparation method for the purpose of gene expression analysis. METHODS: We collected parallel blood samples from healthy donors into the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for 5 days at room temperature and for up to 90 days at 4 and 20 degrees C. Samples were analyzed by Northern blot analysis or reverse transcription-PCR (RT-PCR). RESULTS: Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors. CONCLUSIONS: Compared with whole blood collected in EDTA tubes and extracted by an organic method, the PAXgene Blood RNA System reduced RNA degradation and inhibited or eliminated gene induction in phlebotomy whole blood samples. Storage of whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.