Limited dynamic range of immune response gene expression observed in healthy blood donors using RT-PCR - PubMed (original) (raw)

Clinical Trial

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

Kevin McLoughlin et al. Mol Med. 2006 Jul-Aug.

Abstract

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.

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Figures

Figure 1

Gene distribution across 131 healthy donors. Range of CT values for each gene targeted by the panel of 48 primer sets, across 131 single-time samples. Bars span the range from the 5th to the 95th percentile of CT values for each gene.

Figure 2

Histogram of dynamic ranges of expression values, expressed as fold changes spanning 2 standard deviations of each gene’s ΔCT values (that is, 2−2SD(ΔCT)).

Figure 3

Q-Q normal plots and histograms of ΔCT values for the genes deviating least and most from a normal distribution (IL1R1 in Figure 3A and TNFSF5 in Figure 3B, respectively), according to the Anderson-Darling test. Unit diagonals and normal density curves are drawn on the Q-Q normal plots and histograms, respectively, for comparison with a normal distribution with the same mean and variance as observed. P values computed by the Anderson-Darling normality test are shown above each histogram.

Figure 4

Source of variance in gene expression. (A) Variance components estimated from mixed-effect models, representing variation between subjects (dark grey), between longitudinal samples from same subject (grey), and between replicate RT-PCR reactions for same sample (white). Systematic variations affecting all samples drawn on same date have been subtracted before estimating variance components. (B) Variance components expressed as percentages relative to sum of components.

Figure 5

Time course of expression for 12 genes with significant responses to LPS infusion in 3 healthy male subjects. Whole blood was sampled at pre-LPS (0 h) and 2, 5 and 21 h post-LPS infusion. Gene expression is plotted as ΔCT values relative to mean ΔCT for healthy blood donors, with points and lines colored by subject. Mean and mean ± 2 SD are indicated by horizontal dashed lines. ΔCT scale is inverted, so upward direction corresponds to increasing expression.

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Limited dynamic range of immune response gene expression observed in healthy blood donors using RT-PCR - PubMed (original) (raw)