Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage.
BACKGROUND
Assessment of gene expression is an important component of osteoarthritis (OA) research, greatly improved by the development of quantitative real-time PCR (qPCR). This technique requires normalization for precise results, yet no suitable reference genes have been identified in human articular cartilage. We have examined ten well-known reference genes to determine the most adequate for this application.


RESULTS
Analyses of expression stability in cartilage from 10 patients with hip OA, 8 patients with knee OA and 10 controls without OA were done with classical statistical tests and the software programs geNorm and NormFinder. Results from the three methods of analysis were broadly concordant. Some of the commonly used reference genes, GAPDH, ACTB and 18S RNA, performed poorly in our analysis. In contrast, the rarely used TBP, RPL13A and B2M genes were the best. It was necessary to use together several of these three genes to obtain the best results. The specific combination depended, to some extent, on the type of samples being compared.


CONCLUSIONS
Our results provide a satisfactory set of previously unused reference genes for qPCR in hip and knee OA This confirms the need to evaluate the suitability of reference genes in every tissue and experimental situation before starting the quantitative assessment of gene expression by qPCR.
Pombo-Suarez
Manuel
M
Laboratorio de Investigacion 2 and Rheumatology Unit, Hospital Clinico Universitario de Santiago, Santiago de Compostela, Spain. maposu@mixmail.com
Calaza
Manuel
M
Gomez-Reino
Juan J
JJ
Gonzalez
Antonio
A
D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D002358:Cartilage, Articular / Q000378:metabolism*; D016022:Case-Control Studies; D017931:DNA Primers / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D005819:Genetic Markers; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism; D015207:Osteoarthritis, Hip / Q000235:genetics / Q000378:metabolism; D020370:Osteoarthritis, Knee / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012337:RNA, Ribosomal, 18S / Q000235:genetics / Q000378:metabolism; D012016:Reference Values; D012269:Ribosomal Proteins / Q000235:genetics; D035181:TATA-Box Binding Protein / Q000235:genetics*
D017931:DNA Primers; D005819:Genetic Markers; D012337:RNA, Ribosomal, 18S; C402487:RPL13A protein, human; D012269:Ribosomal Proteins; D035181:TATA-Box Binding Protein; C509817:TBP protein, human; D007041:Hypoxanthine Phosphoribosyltransferase