Letter Regarding Article by Tomic et al, “Transcriptomic and Proteomic Patterns of Systemic Inflammation in On-Pump and Off-Pump Coronary Artery Bypass Grafting”
To the Editor:
We read with interest the article by Tomic and colleagues1 that sought to compare gene expression patterns from leukocytes and protein levels of inflammatory mediators after cardiac surgery. This involved isolation of RNA from blood collected in PaxGene tubes (Qiagen, Valencia, Calif). All subsequent interpretation of gene expression results refers to transcripts isolated from either peripheral blood mononuclear cells (PBMCs) or leukocytes. However, PaxGene tubes allow only for the isolation of RNA from whole blood samples. Centrifugation and removal of a buffy coat layer is necessary to isolate leukocyte-enriched cell populations.
These 2 techniques of isolating RNA have been directly compared.2 The signal-to-noise ratio was found to be significantly higher in the leukocyte-derived samples than in whole blood (P=0.002). Also, the percent present calls identified with the buffy coat method was significantly higher than that identified with the PaxGene method ([mean±SE] 43.2±2.4 versus 28.5±3.7, P<0.0001).
Tomic and colleagues1 conclude that circulating leukocytes are not the source of the inflammatory response because there is discordance between protein- and leukocyte-derived cytokine mRNA. In support of this, a positive control was used to validate the assays by ex vivo stimulation of blood with endotoxin. Subsequent steady-state PBMC transcripts corresponded well to plasma cytokine concentrations.
However, isolation of a PBMC buffy coat layer in the positive control experiment may explain why mRNA and protein levels correlated in this case. Also, the magnitude of the response seen in the positive controls is several log-fold greater than that observed in the patient group, which suggests that ex vivo administration of endotoxin may not be a valid model for the inflammatory response after cardiac surgery. This, combined with the small patient numbers involved, may unfortunately preclude attempts to discern a pattern to the cytokine response.
Although gene expression analyses offer a major step forward in exploring the genome-wide response to inflammatory insults, we have concerns about the reliability of the PaxGene method of isolating RNA.2
Tomic V, Russwurm S, Moller E, Claus RA, Blaess M, Brunkhorst F, Bruegel M, Bode K, Bloos F, Wippermann J, Wahlers T, Deigner H-P, Thiery J, Reinhart K, Bauer M. Transcriptomic and proteomic patterns of systemic inflammation in on-pump and off-pump coronary artery bypass grafting. Circulation. 2005; 112: 2912–2920.
Feezor RJ, Baker HV, Mindrinos M, Hayden D, Tannahill CL, Brownstein BH, Fay A, MacMillan S, Laramie J, Xiao W, Moldawer LL, Cobb JP, Laudanski K, Miller-Graziano CL, Maier RV, Schoenfeld D, Davis RW, Tompkins RG; and the Inflammation and Host Response to Injury, Large-Scale Collaborative Research Program. Whole blood and leukocyte RNA isolation for gene expression analyses. Physiol Genomics. 2004; 19: 247–254.