Comparison of detection techniques for cytokine reverse transcriptase polymerase chain reaction; digoxigenin-labeled polymerase chain reaction permits sensitive detection of cytokine mRNA in rat heart allografts.
The polymerase chain reaction (PCR) is a sensitive method for the analysis of cytokine mRNA expression. The amount of specific mRNA in tissues involved in an inflammatory immune response can be low and therefore requires highly sensitive detection of the PCR products. In our study we have compared different detection techniques in order to replace the commonly used detection by means of radiolabeled probes. Besides the detection of DNA in agarose gels by ethidium bromide (EB), we used detection by digoxigenin (DIG)-labeled probes, as well as the direct incorporation of DIG-labeled nucleotides in the PCR, in comparison to detection by means of 32P-labeled probes. In vitro activated rat lymph node cells, lymph node tissue, and acutely or chronically rejected rat heart allografts were examined for expression of mRNA of the cytokines IL-2 and IFNgamma. The directly DIG-labeled PCR appeared to be the best alternative for detection of PCR products by means of radiolabeled probes. While IL-2 mRNA was not detected by means of EB and IFNgamma mRNA was only detected at the highest PCR cycle numbers in acutely and chronically rejected rat heart allografts, both cytokine mRNA's were readily detected by directly DIG-labeled PCR.
Damoiseaux
J G
JG
Department of Internal Medicine, Faculty of Medicine, Maastricht University, Netherlands.
Theunissen
R
R
Broeren
C P
CP
van Breda Vriesman
P J
PJ
Duijvestijn
A M
AM
D000818:Animals; D015139:Blotting, Southern; D004076:Digoxigenin; D006086:Graft vs Host Disease / Q000175:diagnosis* / Q000276:immunology; D016027:Heart Transplantation / Q000276:immunology*; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics; D008198:Lymph Nodes / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011914:Rats, Inbred BN; D011917:Rats, Inbred Lew; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D014184:Transplantation, Homologous
D007376:Interleukin-2; D012333:RNA, Messenger; D007371:Interferon-gamma; D004076:Digoxigenin