Identification and validation of reference genes for quantitative RT-PCR analysis of retinal pigment epithelium cells under hypoxia and/or hyperglycemia

Liu, Xin; Xie, Jia’nan; Liu, Zaoxia; Gong, Qiang; Tian, Rui; Su, Guanfang

doi:10.1016/j.gene.2016.01.001

Cited by 24 publications

(21 citation statements)

References 29 publications

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“…These differences resulted from the heterogeneous gene expression profile of the tumor type, the experimental conditions, and the stability of the reference genes, being in agreement with the literature (Supplementary Table 1) [21,22,28,29]. In addition, the aforementioned variability can also be attributed to the different algorithms applied by the GeNorm and NormFinder [23,[30][31][32][33]. However, for the single HKG analysis, the first three ranked genes by GeNorm and NormFinder were the same ones, implying no substantial differences in their stability.…”

Section: Discussionsupporting

confidence: 87%

Identification of appropriate housekeeping genes for quantitative RT-PCR analysis in MDA-MB-231 and NCI-H460 human cancer cell lines under hypoxia and serum deprivation

2018

J. Mol. Clin. Med.

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Gene expression studies aimed at analyzing cancer cells under hypoxia and serum deprivation conditions show major potential for understanding molecular mechanisms associated with tumor progression as well as resistance to antitumor agents. To the best of our knowledge, a study for the identification of appropriate housekeeping genes in breast and lung cancer cells under hypoxia and serum deprivation conditions is currently missing. Given the relevance of a reliable and accurate normalization, we herein aimed to identify the appropriate housekeeping genes for breast and lung cancer cell lines cultured under hypoxia and/or serum deprivation. The stability of five commonly used housekeeping genes (ACTB, β 2M, GUSB, 18S rRNA, and PPIA) was assessed after reverse-transcription quantitative real-time PCR in MDA-MB-231 and NCI-H460 cancer cell lines using GeNorm, NormFinder and BestKeeper software. GeNorm and NormFinder ranking revealed ACTB, GUSB and PPIA as the most stable genes for both tumor cell lines. Our results support the use of ACTB/PPIA for MDA-MB-231 and GUSB/PPIA for NCI-H460 cells as the most stable combination for normalization of gene expression under hypoxic and serum deprivation conditions. Our results highlight the importance of the selection of the housekeeping genes in cancer cells subjected to different physiological stresses, such as hypoxia and serum deprivation.

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Section: Discussionsupporting

confidence: 87%

Identification of appropriate housekeeping genes for quantitative RT-PCR analysis in MDA-MB-231 and NCI-H460 human cancer cell lines under hypoxia and serum deprivation

2018

J. Mol. Clin. Med.

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“…PUM1 in HRECs and PPIA in ARPE-19 cells were selected for normalization based on our previous analysis [19, 20]. In the MN osmotic-control groups, SP1 levels did not change significantly; however, when HRECs and ARPE-19 cells were exposed to HG, SP1 levels increased and remained high until days 5 and 7.…”

Section: Resultsmentioning

confidence: 99%

Differentially Expressed MicroRNAs in the Development of Early Diabetic Retinopathy

Gong

Xie

Liu

et al. 2017

Journal of Diabetes Research

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The pathological mechanisms of diabetic retinopathy (DR), a leading cause of blindness in adults with diabetes mellitus, remain incompletely understood. Because microRNAs (miRNAs) represent effective DR therapeutic targets, we identified aberrantly expressed miRNAs associated with cellular dysfunction in early DR and detected their potential targets. We exposed human retinal endothelial cells (HRECs) and a cell line of retinal pigment epithelial (RPE) cells to high glucose (25 mmol/L, 1–7 days) to mimic DR progression and used streptozotocin-injected rats (4–8 weeks) for an in vivo diabetes model. HREC/RPE viability decreased after 24 h incubation and diminished further over 6 days, and Hoechst staining revealed hyperglycemia-induced HREC/RPE apoptosis. Although miR-124/-125b expression decreased with DR progression in vitro and in vivo, miR-135b/-199a levels decreased in retinal cells under hyperglycemia exposure, but increased in diabetic retinas. Moreover, miR-145/-146a expression decreased gradually in high-glucose-treated HRECs, but increased in hyperglycemia-exposed RPE cells and in diabetic rats. Our findings suggested that aberrant miRNA expression could be involved in hyperglycemia-induced retinal-cell dysfunction, and the identified miRNAs might vary in different retinal layers, with expression changes associated with DR development. Therefore, miRNA modulation and the targeting of miRNA effects on transcription factors could represent novel and effective DR-treatment strategies.

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“…Correspondingly, primary human and rat myotubes as well as human embryonic kidney (HEK)293 cells displayed gradually decreasing GAPDH protein content during 24 h serum withdrawal (Pirkmajer and Chibalin 2011). On the other hand, the excess of glucose in culture media (Liu et al 2016; Bakhashab et al 2014) or cell stimulation with growth factors (Tratwal et al 2014) has been demonstrated to affect HKG stability as well.…”

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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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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Identification and validation of reference genes for quantitative RT-PCR analysis of retinal pigment epithelium cells under hypoxia and/or hyperglycemia

Cited by 24 publications

References 29 publications

Identification of appropriate housekeeping genes for quantitative RT-PCR analysis in MDA-MB-231 and NCI-H460 human cancer cell lines under hypoxia and serum deprivation

Identification of appropriate housekeeping genes for quantitative RT-PCR analysis in MDA-MB-231 and NCI-H460 human cancer cell lines under hypoxia and serum deprivation

Differentially Expressed MicroRNAs in the Development of Early Diabetic Retinopathy

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

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