COMPARISON OF DETECTION TECHNIQUES FOR CYTOKINE REVERSE-TRANSCRIPTASEPOLYMERASE-CHAIN-REACTION - DIGOXIGENIN-LABELED POLYMERASE-CHAIN-REACTION PERMITS SENSITIVE DETECTION OF CYTOKINE MESSENGER-RNA IN RAT-HEART ALLOGRAFTS

The polymerase chain reaction (PCR) is a sensitive method for the anal ysis of cytokine mRNA expression. The amount of specific mRNA in tissu es involved in an inflammatory immune response can be low and therefor e requires highly sensitive detection of the PCR products. In our stud y we have compared different detection techniques in order to replace the commonly used detection by means of radiolabeled probes. Besides t he detection of DNA in agarose gels by ethidium bromide (EB), we used detection by digoxigenin (DIG)-labeled probes, as well as the direct i ncorporation of DIG-labeled nucleotides in the PCR, in comparison to d etection by means of P-32 - labeled probes. in vitro activated rat lym ph node cells, lymph node tissue, and acutely or chronically rejected rat heart allografts were examined for expression of mRNA of the cytok ines IL-2 and IFN gamma. The directly DIG-labeled PCR appeared to be t he best alternative for detection of PCR products by means of radiolab eled probes. While IL-2 mRNA was not detected by means of EB and IFN g amma mRNA was only detected at the highest PCR cycle numbers in acutel y and chronically rejected rat heart allografts, both cytokine mRNA's were readily detected by directly DIG-labeled PCR. (C) 1998 Elsevier S cience B.V. All rights reserved.