Purpose: Stability of housekeeping genes as internal reference for RT-qPCR analyses is mandatory for a correct interpretation of results. As no normalization benchmark exists and reference gene validation is highly specific for individual experiments, it was the purpose of this study to identify stable candidates for investigations on periodontal inflammation.
Basic procedures: Human PDL cells from one cell line (Lonza) and three primary donors were challenged with IL-1ß (5 ng/ml) or centrifugation (170 × g) for 6 h under serum-free conditions. Unstimulated cells represented controls. qRT-PCR was performed with a TaqMan® array of 32 housekeeping genes (n = 3). Transcriptional stability was analyzed for (i) mean absolute CT values and (ii) relative fold changes. Finally, stability of mean CT values across specimens was evaluated for most stable candidates. Statistics were performed with one-way ANOVA and Bonferroni correction and one sample t-test, with 95% confidence level. Values represent mean ± SEM.
Main findings: 18S was constant in experimental groups and specimens for mean absolute CT values and relative fold changes, and MT-APT6 for mean absolute CT values. Both genes exhibited low CT thresholds ranging from 20.2 ± 0.1 to 25.9 ± 0.2 for 18S, and from 18.9 ± 0.0-23.7 ± 0.1 for MT-APT6. Likewise stable YWHAZ ranged between 32.6 ± 0.2 and 37.2 ± 0.2 cycles. However, candidates were unstable across specimes.
Principal conclusions: Reference validation is mandatory for RT-qPCR analyses in new experimental designs. Here, only three genes out of 32 turned out to be appropriate candidates. Due to low CT values and stability, 18S and MT-APT6 are most valid genes for data normalization in experiments with PDL cells under inflammatory conditions and are recommended as standards under these premises.
Keywords: Gene expression analysis; Housekeeping genes; Inflammation; Periodontal ligament cells; RT-qPCR.
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