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. 2016 Apr;24(4):719-30.
doi: 10.1016/j.joca.2015.11.007. Epub 2015 Nov 14.

Importance of reference gene selection for articular cartilage mechanobiology studies

A Al-Sabah  1 , P Stadnik  2 , S J Gilbert  3 , V C Duance  4 , E J Blain  5
Affiliations

Importance of reference gene selection for articular cartilage mechanobiology studies

A Al-Sabah et al. Osteoarthritis Cartilage. 2016 Apr.

Abstract

Objective: Identification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively.

Design: The expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder.

Results: Appropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions.

Conclusion: This study demonstrates that commonly used 'reference genes' may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to obtain robust and reproducible qPCR data for analysis/interpretation.

Keywords: Articular cartilage; Chondrocytes; Mechanobiology; Quantitative PCR; Reference genes.

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Figures

Fig. 1
Fig. 1
Expression levels of commonly used reference genes for mRNA normalisation in articular cartilage explants subjected to a loading regime of: [A] 8 MPa (1 Hz, 15 min; N = 3), [B] 5 MPa (1 Hz, 15 min; N = 6) or [C] 2.5 MPa (1 Hz, 15 min; N = 6), all of which were analysed for gene expression 24 h post-cessation of load, [D] 5 MPa (4 Hz, 15 min; N = 6) and gene expression ascertained immediately post-loading, or [E] high-density primary articular chondrocytes subjected to tensile strain (7.5% elongation, 1 Hz, 30 min; N = 2) and gene expression determined 4 h post-cessation of strain. The box and whisker diagrams illustrate the threshold cycles (CT) obtained by qPCR using SYBR® green chemistry and cDNA prepared with 1 μg total RNA.
Fig. 2
Fig. 2
Assessment of gene stability using the comparative delta-Ct method of eight commonly used reference genes in articular cartilage explants either left unloaded or subjected to a loading regime of: [A] 8 MPa (1 Hz, 15 min; N = 3), [B] 5 MPa (1 Hz, 15 min; N = 6) or [C] 2.5 MPa (1 Hz, 15 min; N = 6), all of which were analysed for gene expression 24 h post-cessation of load, [D] 5 MPa (4 Hz, 15 min; N = 6) and gene expression ascertained immediately post-loading, or [E] high-density primary articular chondrocytes subjected to tensile strain (7.5% elongation, 1 Hz, 30 min; N = 2) and gene expression determined 4 h post-cessation of strain. Data is presented as mean threshold cycle (CT) ± standard deviation.
Fig. 3
Fig. 3
Evaluation of reference gene stability using NormFinder in articular cartilage explants either left unloaded or subjected to a loading regime of: [A] 8 MPa (1 Hz, 15 min; N = 3), [B] 5 MPa (1 Hz, 15 min; N = 6) or [C] 2.5 MPa (1 Hz, 15 min; N = 6), all of which were analysed for gene expression 24 h post-cessation of load, [D] 5 MPa (4 Hz, 15 min; N = 6) and gene expression ascertained immediately post-loading, or [E] high-density primary articular chondrocytes subjected to tensile strain (7.5% elongation, 1 Hz, 30 min; N = 2) and gene expression determined 4 h post-cessation of strain.
Fig. 4
Fig. 4
Evaluation of reference gene stability using geNorm in articular cartilage explants either left unloaded or subjected to a loading regime of: [A] 8 MPa (1 Hz, 15 min; N = 3), [B] 5 MPa (1 Hz, 15 min; N = 6) or [C] 2.5 MPa (1 Hz, 15 min; N = 6), all of which were analysed for gene expression 24 h post-cessation of load, [D] 5 MPa (4 Hz, 15 min; N = 6) and gene expression ascertained immediately post-loading, or [E] high-density primary articular chondrocytes subjected to tensile strain (7.5% elongation, 1 Hz, 30 min; N = 2) and gene expression determined 4 h post-cessation of strain.
Fig. 5
Fig. 5
Evaluation of reference gene stability using RefFinder (software that generates its own ranking based on the geometric means of the individual reference genes, having integrated and compared the analyses performed by the individual software packages) in articular cartilage either left unloaded or subjected to a loading regime of: [A] 8 MPa (1 Hz, 15 min; N = 3), [B] 5 MPa (1 Hz, 15 min; N = 6) or [C] 2.5 MPa (1 Hz, 15 min; N = 6), all of which were analysed for gene expression 24 h post-cessation of load, [D] 5 MPa (4 Hz, 15 min; N = 6) and gene expression ascertained immediately post-loading, or [E] high-density primary articular chondrocytes subjected to tensile strain (7.5% elongation, 1 Hz, 30 min; N = 2) and gene expression determined 4 h post-cessation of strain.
Fig. 6
Fig. 6
Effect of reference gene selection on ACAN and MMP3 mRNA levels in cartilage explants either subjected to a [A–D] 5 MPa load (4 Hz, 15 min; N = 6, analysed 24 h post-cessation of load) or a [E–H] 8 MPa load (1 Hz, 15 min; N = 3, analysed 24 h post-cessation of load) using either SDHA, RPL4 and YWHAZ [A, C] or HPRT, RPL4 and YWHAZ [E, G] identified as most stable, or GAPDH and HPRT [B, D] or GAPDH and PPIA [F, H], identified as the least stable reference genes. Data is presented as mean relative fold change ±95% confidence interval normalised to the indicated reference genes and further normalised to the unloaded control explants; statistical analysis was performed using the Student's t-test or Mann–Whitney non-parametric test and statistical significance is indicated.
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