[Comparison of conventional and real-time RT-PCR for the quantitation of jun protooncogene mRNA and analysis of junB mRNA expression in synovial membranes and isolated synovial fibroblasts from rheumatoid arthritis patients].
AP-1 dependent genes, e.g., matrix-metallo-proteinases, are involved in the pathogenesis of rheumatoid arthritis (RA). Therefore, the transcription factor AP-1 and its subunits, proteins of the Jun and Fos proto-oncogene families, are interesting targets for analysis in RA. In this study, we analyzed the mRNA expression of junB in synovial membrane (SM) samples and isolated synovial fibroblasts of patients with RA, osteoarthritis (OA), and normal, non-inflammatory controls. To address the suitability of real-time RT-PCR for the quantitation of Jun proto-oncogene family members, conventional RTPCR and real-time PCR were comparatively applied for junD, a gene representing a major challenge because of its high GC-content (70%, increasing the probability of secondary structures interfering with the PCR) and its sequence homology to other Jun proto-oncogenes. In addition, a comparison was performed concerning the precision, reproducibility, costs, as well as labor and time consumption of the two PCR methods. Real-time RT-PCR proved superior to conventional PCR in terms of precision (mean deviation of measured from employed concentration 58% for real-time PCR vs 225% for conventional PCR), reproducibility, as well as labor and time consumption (4 times less for real-time RT-PCR). Experimental cDNA normalization for equivalent cDNA concentrations by sample dilution was more reliable than mathematical cDNA normalization. However, real-time PCR was 3.6-fold more expensive. Applying the more reliable real-time RT-PCR for the ex vivo analysis of junB mRNA-expression, no significantly different expression of junB was observed in SM or isolated synovial fibroblasts from RA as compared to OA. Interestingly, however, junBmRNA expression was significantly lower in RA SM and borderline significantly lower in OA SM than in normal/non-inflammatory SM, with potential effects on the functional properties of the resulting AP-1 complexes. Immunohistochemical staining of the SM with JunB-specific antibodies showed comparable JunB protein expression in SFB (collagen III mRNA-positive) of RA and OA samples. Thus, real-time RT-PCR appears suitable and time-saving for the quantitation of jun proto-oncogene mRNA-expression in tissue and cell samples with high precision and reproducibility.
Huber
René
R
Experimental Rheumatology Unit, Friedrich Schiller University, Bachstr. 18, 07740 Jena, Germany. Rene.Huber@med.uni-jena.de
Kunisch
Elke
E
Glück
Brigitte
B
Egerer
Renate
R
Sickinger
Stefan
S
Kinne
Raimund W
RW
D001172:Arthritis, Rheumatoid / Q000235:genetics*; D005347:Fibroblasts / Q000737:chemistry*; D005786:Gene Expression Regulation / Q000235:genetics; D016758:Genes, jun / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D000090063:Proto-Oncogene Mas; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics*; D012333:RNA, Messenger / Q000302:isolation & purification; D012016:Reference Values; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013583:Synovial Membrane / Q000737:chemistry*
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger