Automated RNA Extraction by MagNA Pure Followed by Rapid Quantification of Cytokine and Chemokine Gene Expression with Use of Fluorescence Resonance Energy Transfer

Cytokines and chemokines, a superfamily of small cytokine-like molecules, are key mediators of immunity and inflammation. Detection of cytokines and chemokines in disease states provides useful diagnostic tools for investigating host responses to invading organisms, tumors, and trauma.Different reverse transcription-PCR (RT-PCR) protocols have been described in the literature, including semiquantitative, quantitative, and competitive PCR techniques, but most of them were labor-intensive and time-consuming (1)(2). Recently, real-time PCR assays have become a major tool for quantifying the number of DNA or cDNA copies in different settings and clinical materials. These assays offer a standardized, rapid, accurate, and reproducible method that combines rapid in vitro amplification with real-time quantification of the DNA or cDNA load. However, to date, only limited data are available for quantification of cytokine gene expression (3)(4). In addition, there is a need for rapid, sensitive, reliable, and standardized methods for RNA extraction.Here we present an automated standardized protocol for the extraction of RNA from human peripheral blood mononuclear cells (PBMCs), using the MagNA Pure LC system (Roche Diagnostics), combined with a rapid and easy-to-perform quantitative PCR assay, using the LightCycler (Roche Diagnostics) amplification and detection system. We applied this protocol to quantification of mRNA expression of the following immunorelevant genes: interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-18, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), macrophage-inducing protein-3α (MIP-3α), SCYB-11 (IFN-inducible T-cell-α chemoattractant; H174; CXCL11), intracellular adhesion molecule-1 (ICAM-1; CD54), and nuclear factor-κB (NFκB), the transcription factor and central mediator of immune response.PBMCs were prepared from 50 mL of heparinized fresh venous blood obtained from healthy donors (n = 30). PBMCs were separated by centrifugation over Ficoll and resuspended in 1 mL of phosphate-buffered saline. The PBMC concentration was adjusted to 1 × 106/mL in RPMI 1640 containing 100 …