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Technical Briefs |
Department of Experimental and Clinical Pharmacology and Toxicology, Emil-Fischer-Center, University of Erlangen-Nuremberg, Fahrstrasse 17, D-91054 Erlangen, Germany;
aauthor for correspondence: fax 49-9131-22774, e-mail pahl{at}pharmakologie.uni-erlangen.de
Emerging technologies such as quantitative, real-time reverse transcription-PCR (RT-PCR) and microarrays enable the accurate quantification of ribonucleic acids in clinical samples. Gene expression profiling of tumor samples with use of microarray technology has become the prototypic application of this new type of molecular diagnostics. DNA microarray analysis of different closely related tumor types revealed that this technology is able to distinguish different tumors (1).
The concentrations of specific mRNAs, however, may change between specimen acquisition and the beginning of analysis, and these changes are not well understood. When blood is studied, specimen collection and sample preparation techniques by themselves may produce changes in gene expression ex vivo, e.g., it has been shown that anticoagulants cause ex vivo changes in cytokine production (2).
If changes in gene expression occur after phlebotomy, the valid interpretation of basal mRNA expression data as sensitive markers in clinical studies requires the standardization of conditions for assaying patient blood samples. The current study was undertaken to analyze changes in gene expression in human peripheral blood stored ex vivo. We studied ex vivo changes in expression of several genes by use of quantitative, real-time RT-PCR and evaluated procedures for standardized blood sampling for molecular diagnostics.
After receiving informed consent, we obtained blood from healthy donors (23–60 years of age). Whole blood was collected with use of the VACUTAINER® Safety-LokTM Blood Collection Set and either PAXgeneTM Blood RNA Tubes (2.5 mL draw volume; PreAnalytiX) or EDTA tubes (VACUTAINER PLUSTM 6-mL dipotassium EDTA tubes). This investigation was performed according to the International Declarations of Helsinki and Tokyo. After phlebotomy, PAXgene Blood RNA Tubes were stored at room temperature until processing. Alternatively, immediately after phlebotomy, 1 mL of water and 6 mL of Trizol LS reagent (Invitrogen) were added to 1 mL of whole blood collected in EDTA tubes and stored at room temperature until processing. Blood was mixed with the reagent and incubated at room temperature until processing. To study ex vivo changes in gene expression, whole blood collected in EDTA tubes was stored under sterile conditions at room temperature and then processed using Trizol LS. Microbial contamination was excluded by sterility testing.
RNA was prepared from blood collected in PAXgene tubes with use of the PAXgene Blood RNA Kit (PreAnalytiX) according to the manufacturer’s instructions, including DNase treatment. RNA was prepared by use of Trizol LS according to the manufacturer’s instruction (Invitrogen). RT-PCR was performed with the QIAGEN OneStep RT-PCR Kit (QIAGEN) and gene-specific primers. Quantitative, real-time RT-PCR was performed with TaqMan assays on an ABI PRISM 7700 or 7900 instrument. Primers and probes were synthesized by TIB Molbiol. Sequences are available with the online version of this Technical Brief athttp://www.clinchem.org/content/vol48/issue12/. The amount of mRNA was calculated by the 
CT method (3).
We first detected changes in concentrations of transcript of the proinflammatory cytokine tumor necrosis factor-
(TNF), which is known to be sensitive to extracellular stimuli in whole blood after phlebotomy (Fig. 1A
). TNF transcript increased rapidly up to 20-fold compared with concentrations at time of phlebotomy.
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As shown in Figs. 1 and 2 in the Data Supplement (available with the online version of this Technical Brief athttp://www.clinchem.org/content/vol48/issue12/), a general RNA degradation in whole blood occurs ex vivo over time. We compared different normalization methods for study of TNF expression. Despite normalization to two different mRNAs (ß-actin and glyceraldehyde 3-phosphate dehydrogenase), to 18S rRNA, and to the total RNA concentration, these four normalization methods did not generate significantly different results (Fig. 3 in the Data Supplement).
We looked for methods of preserving transcript concentrations after blood collection. We first tried addition of a mixture of acidic phenol and guanidine isothiocyanate to blood immediately after collection (4). We next used the PAXgene Blood RNA Tube, a blood collection tube containing an additive that stabilizes cellular RNA. We measured TNF mRNA and compared whole blood samples collected in conventional EDTA tubes with samples treated as described above (Fig. 1A
). All samples were stored at room temperature. Both stabilization methods preserved the transcript concentrations of all TNF for the entire observation period. No significant changes in TNF mRNA concentrations were observed. However, immediate addition of a toxic solution to blood is impracticable in the clinic. The use of an integrated collection device containing stabilizing reagents seems preferable.
We hypothesized that these expression changes result from ongoing or induced transcription in cells in whole blood. To address this question, we added a transcription inhibitor, actinomycin D, at different times to the blood and measured TNF mRNA (Fig. 1B
). The PAXgene Blood RNA system was used for stabilization. Again, only small changes in expression were observed with this method compared with untreated samples. When actinomycin was added to whole blood collected in EDTA tubes immediately after phlebotomy, the increase of TNF mRNA measured after 1, 3, and 7 days was abolished (Fig. 1B
). Furthermore, adding actinomycin at days 1 or 3 and measuring mRNA at day 7 revealed time-dependent inhibition of cytokine induction. This indicates that stimuli that induce transcription are active only for the first 24 h after phlebotomy. After that time, cells are adapted and no further changes occur. The change of environment after phlebotomy is likely to affect gene expression. Adherence to the plastic walls of standard blood collection tubes may be one such stimulus, as this has been shown to induce proinflammatory cytokines (5).
We investigated whether expression of other inflammatory genes was also affected during storage of whole blood (Table 1
). Although the expression of interleukin-6 (IL-6) and TNF increased up to 30-fold, IL-1ß decreased slightly after collection.
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The balance of subsets of T-helper (Th) cells affects the outcome of immunologic diseases (6). The balance of Th1 and Th2 cells is measured by the ratio of interferon-
(IFN) and IL-4. We observed increased IFN transcripts, whereas IL-4 transcript concentrations hardly changed (Table 1
). Measurement of these transcripts in unstabilized samples would therefore give a false ratio of these cytokines that did not reflect the ratios in vivo, possibly leading to incorrect interpretation of a patient’s immunologic status. IL-10 is a suppressive cytokine produced by various cell types such as monocytes or regulatory T cells (7), and its transcripts were also increased after 24 h (Table 1
). Chemokines are a group of cytokines functionally characterized by their ability to attract other cells, causing cell migration (8). We analyzed mRNAs of three typical chemokines known to be inducible in certain cell types: RANTES, Mip1, and eotaxin. Expression of all chemokines increased after blood collection (Table 1
).
As we found that transcriptional processes were responsible for the observed changes, we analyzed transcription factor nuclear factor-
B (NF-B). This transcription factor induces a variety of immediate early genes, including most of the genes analyzed in the present study (9). In quiescent cells, NF-B is inactive because of association with inhibitor B (IB) proteins. On inflammatory activation, IB molecules are degraded by the proteasome. One of the hallmarks of NF-B activation is rapid induction of IB mRNA synthesis, which leads to the renewed synthesis of its own inhibitor, producing a feedback inhibition (10). IB mRNA synthesis therefore can be used as a surrogate marker of NF-B activation. Indeed, we found a marked increase in IB mRNA in unstabilized blood (Table 1
). This suggests that NF-B makes a major contribution to increased mRNA concentrations after phlebotomy.
In summary, we found that transcripts of 10 genes changed in blood after phlebotomy. No general pattern could be observed. Both the PAXgene Blood RNA system and actinomycin pretreatment blunted changes for most mRNAs. Interestingly, actinomycin treatment did not inhibit induction of eotaxin. Therefore, other mechanisms must account for the observed induction. In the absence of nucleic acid stabilization, artifactual results may be produced. Standard protocols are needed for study of gene expression profiles in clinical samples. These procedures must contain methods to standardize and stabilize clinical samples before RNA isolation and analysis.
Acknowledgments
We thank Isabella Kolberg and Petra Stöcker for excellent technical assistance and Stewart Jurgensen for assistance with Northern blots. This work was supported by the Bundesministerium für Bildung und Forschung (Grant 312242).
References
B in cytokine gene regulation. Am J Respir Cell Mol Biol 1997;17:3-9.B regulates IB by two distinct mechanisms. Genes Dev 1993;7:1266-1276.The following articles in journals at HighWire Press have cited this article:
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  M. Cankovic, R. C. Varney, L. Whiteley, R. Brown, R. D'Angelo, D. Chitale, and R. J. Zarbo The Henry Ford Production System: LEAN Process Redesign Improves Service in the Molecular Diagnostic Laboratory: A Paper from the 2008 William Beaumont Hospital Symposium on Molecular Pathology J. Mol. Diagn., September 1, 2009; 11(5): 390 - 399. [Abstract] [Full Text] [PDF]
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 J.-B. Marteau, S. Mohr, M. Pfister, and S. Visvikis-Siest Collection and Storage of Human Blood Cells for mRNA Expression Profiling: A 15-Month Stability Study Clin. Chem., July 1, 2005; 51(7): 1250 - 1252. [Full Text] [PDF]
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