Evaluation of reference genes for human chondrocytes cultured in several different thermal environments

Ito, Akira; Aoyama, Tomoki; Tajino, Junichi; Nagai, Masaya; Yamaguchi, Satoshi; Iijima, Hirotaka; Zhang, Xiangkai; Akiyama, Haruhiko; Kuroki, Hiroshi

doi:10.3109/02656736.2014.906048

Cited by 6 publications

(7 citation statements)

References 29 publications

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“…Analyzing HKG in colon and liver tissues from CRC patients with hepatic metastases, Blanquicett et al (2002) observed that ribosomal HKG displayed the most stable expression while those involved in metabolic pathways were the least stable ones. Substantiating the notion, we and others (Dydensborg et al 2006; Bakhashab et al 2014; Jacobsen et al 2014) demonstrated superior stability of RPLP0 and Bian et al (2015), Powell et al (2014), and Ito et al (2014) that of another ribosomal protein— RPL13A . In turn, PGK1 was one of the least stable genes in our study, although GAPDH , encoding an enzyme involved in the same metabolic pathway, performed well.…”

Section: Discussionmentioning

confidence: 59%

“…Yet, with stability of its expression being suboptimal, it could affect the statistical outcome. Of note, preservation of GAPDH expression upon altered conditions has been reported for human umbical vein endothelial cells (HUVECs) grown under hyperglycemic conditions (Bakhashab et al 2014), chondrocytes cultured at different temperatures (Ito et al 2014) or blood cells subjected to radiation (Vaiphei et al 2015). …”

Section: Discussionmentioning

confidence: 99%

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Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

et al. 2016

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Careful selection of housekeeping genes (HKG) is prerequisite to yield sound qPCR results. HKG expression varies in response to hypoxia but the effect of manipulations of serum availability, a common experimental procedure, remains unknown. Also, no data on HKG expression stability across colon adenocarcinoma lines that would aid selection of normalizers suitable for studies involving several lines are available. Thus, we evaluated the effect of serum availability on the expression of commonly used HKG (ACTB, B2M, GAPDH, GUSB, HPRT1, IPO8, MRPL19, PGK1, PPIA, RPLP0, RPS23, SDHA, TBP, UBC, and YWHAZ) in seven colon adenocarcinoma cell lines (Caco-2, DLD-1, HCT116, HT29, Lovo, SW480, and SW620). Sets of stably expressed line-specific and pan-line HKG were validated against absolutely quantified CDKN1A, TP53, and MDK transcripts. Both serum availability and line type affected HKG expression. UBC was fourfold down-regulated and HPRT1 1.75-fold up-regulated in re-fed HT29 cultures. Line-to-line variability in HKG expression was more pronounced than that caused by altering serum availability and could be found even between isogenic cell lines. PPIA, RPLP0, YWHAZ, and IPO8 were repeatedly highly ranked while ACTB, B2M, UBC, and PGK1 were ranked poorly. Normalization against PPIA/RPLP0/SDHA was found optimal for studies involving various colon adenocarcinoma cell lines subjected to manipulations of serum availability. We found HKG expression to vary, more pronouncedly by line type than growth conditions with significant differences also between isogenic cell lines. Although using line-specific normalizers remains optimal, a set of pan-line HKG that yields good estimation of relative expression of target genes was proposed.

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Section: Discussionmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

et al. 2016

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“…The data obtained by real-time PCR were analyzed by the comparative threshold cycle method. Briefly, the target gene quantity was normalized to that of RPL13a and YWHAZ , which are known to be stable under different thermal environments [ 37 ]. The mean value of the calibration sample (the cells cultured in monolayer at 32°C) was set to 1 and the values obtained for each of the other conditions were calculated relative to that of the calibration sample.…”

Section: Methodsmentioning

confidence: 99%

Culture Temperature Affects Human Chondrocyte Messenger RNA Expression in Monolayer and Pellet Culture Systems

et al. 2015

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Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C) for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and citrate synthase (CS), which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1) and aggrecan (ACAN), was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y)-box 9 (SOX9), which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and chondrogenesis.

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“…The data obtained by qRT‐PCR were analyzed using the comparative threshold cycle method. Briefly, the quantity of the target genes was normalized to the expression levels of RPL13a and YWHAZ , which have been proven to be stable under different thermal conditions . The value of the calibration sample (cells cultured at 32°C on day 3) was set to 1, and the values for the other conditions were reported relative to that of the calibration sample.…”

Section: Methodsmentioning

confidence: 99%

Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes

Ito

Aoyama

Iijima

et al. 2015

Journal Orthopaedic Research

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To date, there have been few studies on how temperature affects the phenotype and metabolism of human chondrocytes. Thus, the purpose of this study was to elucidate the effects of culture temperature on chondrocyte redifferentiation and extracellular matrix (ECM) formation using dedifferentiated mature human chondrocytes in vitro. Dedifferentiated chondrocytes were cultured in a pellet culture system for up to 21 days. The pellets were randomly divided into three groups with different culture temperature (32, 37, and 41˚C). Chondrocyte redifferentiation and ECM formation were evaluated by wet weight, messenger ribonucleic acid (mRNA), histological, and biochemical analyses. The results showed that the wet weight and the mRNA expressions of collagen type II A1 and cartilage oligomeric matrix protein at 37˚C were higher than the corresponding values at 32˚C. The histological and biochemical analyses revealed that the syntheses of type II collagen and proteoglycan were promoted at 37˚C compared to those at 32˚C, whereas they were considerably inhibited at 41˚C. In conclusion, the results obtained herein indicated that temperature affects chondrocyte redifferentiation and ECM formation, and modulation of temperature might thus represent an advantageous means to regulate the phenotype and biosynthetic activity of chondrocytes. ß

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Evaluation of reference genes for human chondrocytes cultured in several different thermal environments

Abstract: The combination of RPL13a and YWHAZ might be suitable as reference genes for human chondrocytes cultured at 32 °C, 37 °C, and 41 °C in monolayer, pellet, or combined cultures.

Cited by 6 publications

References 29 publications

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

Culture Temperature Affects Human Chondrocyte Messenger RNA Expression in Monolayer and Pellet Culture Systems

Culture temperature affects redifferentiation and cartilaginous extracellular matrix formation in dedifferentiated human chondrocytes

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