Stability of NT-proBNP in serum specimens collected in Becton Dickinson Vacutainer (SST) tubes.

reaction is 2 ␮L). If probes are labeled with two different dyes (LCRed640 and LCRed705), two independent 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 populations. 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 concentrations 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 compensate 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, standardized 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 degradation and allows several tests to be performed within 1 day. Furthermore, reports describing other RT-PCR assays 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. However , 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. Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase …