Cycler reaction is 2 L). If probes are labeled with two different dyes (LCRed640 and LCRed705), two indepen- dent genes can be quantified in a single reaction, doubling the number of targets. Other methods, such as intracellu- lar cytokine staining, require at least 10 6 cells for the quantification of only one cytokine. Thus the proposed method enables determination of different subpopula- tions of cells, e.g., precursor frequencies of T-cell sub- populations, or maturation states of defined cell popula- tions. Finally, the method also provides an efficient tool to confirm selected data from gene array experiments. Because of poor interassay reproducibility, cytokine concentrations were expressed in relative copy numbers. We thus achieved appropriate results when cDNA con- centrations were measured spectrophotometrically before reverse transcription and PCR and when strong stimula- tors such as PHA and LPS were used (Fig. 1B). However, for weak stimuli, an internal control is needed to compen- sate for minor variations in the RNA concentration. This can be fulfilled by normalization against a housekeeping gene or ribosomal RNA. Recently, several reports have been published that describe quantification of cytokine mRNA by real-time RT-PCR (4, 7–9 ). All of these protocols rely on manual RNA extraction by spin column technology [QIAamp Blood Kit (7)] or High Pure RNA isolation (8). However, to achieve a high sensitivity for the RT-PCR assay, stan- dardized RNA extraction protocols with high-quality nucleic acid purification are mandatory. Our proposed protocol uses a fully automated assay with commercially available reagents, which reduces the risk of RNA degra- dation and allows several tests to be performed within 1 day. Furthermore, reports describing other RT-PCR as- says have been limited to the detection of simian cytokine RNA (9) or describe the detection of only a very limited number of target genes (4, 7). Autschbach et al. (8) described a LightCycler-based assay for the detection of 35 mRNAs encoding cytokines and chemokines to define mediator profiles in inflammatory bowel disease. How- ever, this protocol relies on the SYBR Green I detection format, which is less sequence specific than the hybrid- ization probes format and requires additional melting curve analysis to exclude primer-dimers. In conclusion, the proposed method for analysis of cytokine and chemokine gene expression is accurate, reliable, fast, and easy to perform and may help to prevent graft failure and reduce patient morbidity and mortality. We thank O. Landt (TIB Molbiol, Berlin, Germany) for excellent technical support. This project was supported by Grants 01GS0114IES16T031 and 01GS0114IES16T051 from the Nationales Genomforschungsnetz (NGFN). References 1. Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A 1990;87:2725–9. 2. O’Garra A, Vieira P. Polymerase chain reaction for detection of cytokine gene expression. Curr Opin Immunol 1992;4:211–5. 3. Overbergh L, Valckx D, Waer M, Mathieu C. Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 1999;11:305–12. 4. Stordeur P, Poulin LF, Craciun L, Zhou L, Schandene ´ L, de Lavareille A, et al. Cytokine mRNA quantification by real-time PCR. J Immunol Methods 2002; 259:55– 64. 5. Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ. The LightCycler: a microvolume multisample fluorimeter with rapid temperature. Biotechniques 1997;22:176 – 81. 6. Benveniste O, Martin M, Villinger F, Dormont D. Techniques for quantification of mRNAs. Cytokines Cell Mol Ther 1998;4:207–14. 7. Ku ¨hne BS, Oschmann P. Quantitative real-time RT-PCR using hybridization probes and imported standard curves for cytokine gene expression analysis. Biotechniques 2002;33:1078 – 89. 8. Autschbach F, Giese T, Gassler N, Sido B, Heuschen G, Heuschen U, et al. Cytokine/chemokine messenger-RNA expression profiles in ulcerative colitis and Crohn’s disease. Virchows Arch 2002;441:500 –13. 9. Hofmann-Lehmann R, Williams AL, Swenerton RK, Li PL, Rasmussen RA, Chenine AL, et al. Quantitation of simian cytokine and -chemokine mRNAs using real-time reverse transcriptase-polymerase chain reaction variations in expression during chronic primate lentivirus infection. AIDS Res Hum Retro- viruses 2002;18:627–39. Stability of NT-proBNP in Serum Specimens Collected in Becton Dickinson Vacutainer (SST) Tubes, Amitava Dasgupta, 1* Loretta Chow, 2 Gertie Tso, 2 and Lupe Nazareno 2 ( 1 Department of Pathology and Laboratory Medicine, University of Texas-Houston Medical School, 6431 Fan- nin, MSB 2.292, Houston, TX 77030; 2 Memorial-Hermann Laboratory Services, Houston, TX 77030; * author for cor- respondence: fax 713-500-0730, e-mail Amitava.Dasgupta @uth.tmc.edu) Atrial natriuretic peptides, B-type natriuretic peptide (BNP), and C-type peptides play important roles in the cardiovascular system (1–3 ). ProBNP is a 108-amino acid peptide that is secreted by the ventricle and is cleaved to physiologically active BNP (amino acids 77–108) and N-terminal fragment NT-proBNP (amino acids 1–76) (3). NT-proBNP is a useful marker for heart failure, including congestive heart failure (4–6). Recently, Roche Diagnos- tics marketed an automated NT-proBNP assay on the Elecsys analyzer. The assay uses polyclonal antibodies against NT-proBNP. The concentration of NT-proBNP in healthy individuals depends on both age and gender, with females and elderly patients having higher concen- trations. The clinical sensitivity and specificity of NT- proBNP at a cutoff of 125 ng/L for male patients younger than 75 years are 81.6 –91.7% (sensitivity) and 86.7–95.7% (specificity), depending on age. The corresponding sensi- tivity and specificity for female patients are 86.7–94.3% (sensitivity) and 57.8 – 84.9% (specificity), respectively. The negative predictive value of this test is almost 100% at a clinical threshold of 125 ng/L, according to the package insert (NT-proBNP; Roche Diagnostics). A common practice in clinical laboratories is to draw blood in serum-separator tubes because of the advantage of the barrier gel, which facilitates rapid separation of serum from cellular constituents of blood and prevents hemolysis during prolonged storage. However, measured 958 Technical Briefs