Quantitative biomarker analysis of synovial gene expression by real-time PCR.
Synovial biomarker analysis in rheumatoid arthritis can be used to evaluate drug effect in clinical trials of novel therapeutic agents. Previous studies of synovial gene expression for these studies have mainly relied on histological methods including immunohistochemistry and in situ hybridization. To increase the reliability of mRNA measurements on small synovial tissue samples, we developed and validated real time quantitative PCR (Q-PCR) methods on biopsy specimens. RNA was isolated from synovial tissue and cDNA was prepared. Cell-based standards were prepared from mitogen-stimulated peripheral blood mononuclear cells. Real time PCR was performed using TaqMan chemistry to quantify gene expression relative to the cell-based standard. Application of the cellular standard curve method markedly reduced intra- and inter-assay variability and corrected amplification efficiency errors compared with the C(t) method. The inter-assay coefficient of variation was less than 25% over time. Q-PCR methods were validated by demonstrating increased expression of IL-1beta and IL-6 expression in rheumatoid arthritis synovial samples compared with osteoarthritis synovium. Based on determinations of sampling error and coefficient of variation, twofold differences in gene expression in serial biopsies can be detected by assaying approximately six synovial tissue biopsies from 8 to 10 patients. These data indicate that Q-PCR is a reliable method for determining relative gene expression in small synovial tissue specimens. The technique can potentially be used in serial biopsy studies to provide insights into mechanism of action and therapeutic effect of new anti-inflammatory agents.
Boyle
David L
DL
Center for Innovative Therapy, Division of Rheumatology, Allergy and Immunology, UCSD School of Medicine, La Jolla, California, USA. dboyle@ucsd.edu
Rosengren
Sanna
S
Bugbee
William
W
Kavanaugh
Arthur
A
Firestein
Gary S
GS
D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001172:Arthritis, Rheumatoid / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015415:Biomarkers; D001706:Biopsy; D002138:Calibration; D003199:Computer Systems; D018076:DNA, Complementary / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D013583:Synovial Membrane / Q000378:metabolism* / Q000473:pathology
D000893:Anti-Inflammatory Agents; D015415:Biomarkers; D018076:DNA, Complementary; D011506:Proteins; D012333:RNA, Messenger