Protocol: Analysis of brain mRNA by reverse transcription-polymerase chain reaction and hybridization with digoxigenin-labeled DNA probe

The interleukin-1 family of polypeptides (IL-1 alpha , IL-1 beta and IL-1 receptor antagonist (RA)) induces various centrally mediated host defense responses to infectious pathogens [3]. Considerable interest has focussed on IL-1 as a mediator in disease and in the production of systemic acute phase responses. We have recently studied the effects of a peripheral stimulation by intraperitoneal (i.p.) administration of lipoposaccharide (LPS) on the mRNAs expressions of IL-1 ( alpha , beta , RA) and their receptors (IL-1 receptor type I and type II (IL-1R1, IL-1R2)) in the central nervous system (CNS) [5, 6]. The levels of these expressions being very low in the CNS, the reverse transcription-polymerase chain reaction (RT-PCR) techniques are required for these studies. RT-PCR is a developed method of identifying mRNAs in very small amount of nucleic acid. We have previously developed a method to choose specific PCR primers [9]. The detection of specific PCR products is extremely important. Since amplifications with these specific PCR primers can be achieved under the same conditions (buffers and temperatures) reliable results can be obtained. Characterization of a PCR product requires the use of a specific DNA probe that hybridizes to the region of interest. In addition to providing specificity of detection, the use of labeled DNA probes provides increased sensitivity over ethidium bromide staining. We have previously described a method of synthesis of non-radioactive probe labeled with digoxigenin by nested PCR [5, 6, 8]. Moreover the major advantage to the use of non-radioactive label is that it does not have a short half-life and can last for weeks or even months. A quantification of the PCR products can be obtained using a method based on the analysis of photographic negatives of agarose gels. (c) 1997 Elsevier Science B.V. All rights reserved.