Novel multi-probe RNase protection assay (RPA) sets for the detection of murine chemokine gene expression

Chemokines play an essential role in immune and inflammatory reactions via the recruitment of leukocytes. Studying the role of chemokines in vivo is c omplicated by the redundancy of their action and by their promiscuous recep tor usage. The simultaneous analysis of several chemokines is, therefore, a dvantageous in order to obtain a comprehensive view of chemokine participat ion in inflammatory and infectious processes. At present, no multi-probe de tection systems are available fur the analysis of recently described chemok ines. In this study, new multi-probe RNase protection assay (RPA) template sets were developed for the analysis of murine chemokines. Chemokine cDNA f ragments were generated by RT-PCR and individually subcloned into the plasm id pGEM-T providing a T7 promotor. In this way, two multi-probe template se ts were constructed each containing six chemokine sequences (CXCL12/SDF-1, XCL1/lymphotactin, CCL20/exodus-1, CCL25/TECK, CX3CL1/fractalkine, CXCL1/KC , and CCL20/MDC, CXCL9/MIG, CCL9/10/MIP-1 gamma, CXCL13/BLC, CCL12/MCP-5, C CL19/ELC, respectively) and templates for the two house-beeping genes L32 a nd GAPDH. The evaluation of these RPA template sets in various murine model s demonstrated their suitability for the analysis of the above chemokines b oth under constitutive and infection-induced conditions. To reduce the pers onal radiation hazard, we found that P-32 could be replaced by P-33 without any loss of assay-sensitivity. These new RPA multi-probe sets provide valu able tools for the simultaneous quantitative determination of gene expressi on of multiple murine chemokines of both constitutive and inducible type. ( C) 2001 Elsevier Science BN. All rights reserved.