Response to Letter Regarding Article by Tomic et al, “Transcriptomic and Proteomic Patterns of Systemic Inflammation in On-Pump and Off-Pump Coronary Artery Bypass Grafting”
We thank Dr O’Dwyer and colleagues for their letter in which they raise concerns regarding interpretation of the discordance between protein and cytokine mRNA levels in patients undergoing coronary bypass surgery and regarding the isolation of RNA from whole blood by the PaxGene methodology.1 We would like to use this opportunity to clarify that RNA was isolated for all experiments by the PaxGene whole blood method, including the positive control, ie, ex vivo endotoxin-spiked whole blood cultures. We agree that the legend to Figure 3 in our original article is somewhat misleading because we refer to “total RNA isolated from [peripheral blood mononuclear cells],” although as is increasingly acknowledged, RNA might also be derived from sources other than peripheral blood mononuclear cells exclusively.2,3
Preparation of RNA for microarrays is indeed a critical preanalytical step, as differences attributable to the mode of RNA isolation might even outweigh changes in transcript expression caused by a biological insult, as shown by the Glue Grant consortium.4 Nevertheless, although there are changes that clearly depend on the method of RNA preparation, the question remains which of the established methods may best reflect the cell response in vivo and yield the de facto transcription pattern. Although PaxGene offers a number of technical advantages, increased noise and reduced responsiveness reflect possible limitations. However, immediate-early gene response during and after isolation are likely to result in false-positive results for all protocols that involve cell enrichment. Furthermore, induction of genes on density gradient centrifugation or magnetic sorting, for example, might depend on the functional state of leukocytes, and thus would be different for naïve as opposed to primed leukocytes. Therefore, RNA isolation for reliable transcriptional profiling will likely be plagued by the lack of an accepted methodology for the near future. On the basis of our recent results in stabilized whole blood, it will be a challenge to investigate gene expression in subclasses of leukocytes and in platelets from patients undergoing coronary bypass surgery.
Tomic V, Rußwurm S, Moller E, Claus RA, Blaess M, Brunkhorst F, Bruegel M, Bode K, Bloos F, Wippermann J, Wahlers T, Deigner HP, 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.
Jozsef L, Khreiss T, El Kebir D, Filep JG. Activation of TLR-9 induces IL-8 secretion through peroxynitrite signaling in human neutrophils. J Immunol. 2006; 176: 1195–1202.
Gnatenko DV, Dunn JJ, McCorkle SR, Weissmann D, Perrotta PL, Bahou WF. Transcript profiling of human platelets using microarray and serial analysis of gene expression. Blood. 2003; 101: 2285–2293.
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; 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.