title: Stability of housekeeping genes in human intervertebral disc, endplate and articular cartilage cells in multiple conditions for reliable transcriptional analysis.
abstract: Quantitative gene expression analysis is widely used to evaluate the expression of specific tissue markers. To obtain reliable data it is essential to select stable housekeeping genes whose expression is not influenced by the anatomical origin of cells or by the culture conditions. No studies have evaluated housekeeping gene stability in intervertebral disc (IVD) cells and only few studies using cartilaginous endplate (CEP) and articular cartilage (AC) cells are present in the literature. We analysed the stability of four candidate housekeeping genes (GAPDH, TBP, YWHAZ and RPL13A) in human cells isolated from nucleus pulposus (NP) and annulus fibrosus (AF), CEP and AC. Cell isolation, expansion, cryoconservation, and differentiation in 3D pellets were tested. GeNorm, NormFinder, BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability. In each cell population, TBP alone or combined with YWHAZ was identified as the best normaliser in both monolayer and 3D pellets. GAPDH was the best performer only for AC cells in monolayer. In most culture conditions considering groups of two or more cell types, TBP was the most stable and YWHAZ was the second choice. GAPDH was the best performer only in 3D pellets with factors for AC and AF combined with CEP cells. RPL13A was the most stable only for AF with CEP cells at isolation. Our findings will be useful to properly design the experimental set-up of studies involving IVD, CEP or AC cells in different culture conditions, in order to obtain accurate and high quality data from quantitative gene expression analysis.
authors.affiliation: Laboratory of Experimental Biochemistry and Molecular Biology, IRCCS Galeazzi Orthopaedic Institute, Via R Galeazzi 4, 20161 Milan, Italy.alessandra.colombini@grupposandonato.it.
authors.forename: S
authors.initials: S
authors.lastname: Lopa
authors.forename: C
authors.initials: C
authors.lastname: Ceriani
authors.forename: R
authors.initials: R
authors.lastname: Cecchinato
authors.forename: L
authors.initials: L
authors.lastname: Zagra
authors.forename: M
authors.initials: M
authors.lastname: Moretti
authors.forename: A
authors.initials: A
authors.lastname: Colombini
mesh_terms: D000368:Aged; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism*; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D020043:Genes, Essential; D056726:Genetic Association Studies; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism*; D015203:Reproducibility of Results; D059010:Single-Cell Analysis