RPL13A as a reference gene for normalizing mRNA transcription of ovarian cancer cells with paclitaxel and 10-hydroxycamptothecin treatments

Bian, Zehua; Ye, Yu; Quan, Chao; Guan, Rongwei; Jin, Yan; Wu, Jie; Xu, Lei; Chen, Feng; Bai, Jing; Sun, Wenjing; Fu, Songbin

doi:10.3892/mmr.2014.3108

Cited by 27 publications

(22 citation statements)

References 26 publications

(27 reference statements)

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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%

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%

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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“…In order to get reliable results from RT-PCR research, it is suggested to combine and use two or more reference genes. The geNorm software also calculates the optimal number of reference genes combined to use for normalization based on a pair wise variation (Vn/(n+1)) analysis [22, 28]. …”

Section: Resultsmentioning

confidence: 99%

“…Thus it is necessary to identify stable internal reference genes in different experiment settings. Recently, it is reported that B2M and 18S RNA are suitable internal reference genes in serum stimulated samples [19]; GUSB and PPIA are believed to serve as internal reference genes in ovarian cancer [33], while RPL13A is identified to stably transcript in ovarian cancer cells treated with PTX and HCPT [28]. …”

Section: Discussionmentioning

confidence: 99%

Identification of TMEM208 and PQLC2 as reference genes for normalizing mRNA expression in colorectal cancer treated with aspirin

et al. 2017

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Numerous evidences indicate that aspirin usage causes a significant reduction in colorectal cancer. However, the molecular mechanisms about aspirin preventing colon cancer are largely unknown. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a most frequently used method to identify the target molecules regulated by certain compound. However, this method needs stable internal reference genes to analyze the expression change of the targets. In this study, the transcriptional stabilities of several traditional reference genes were evaluated in colon cancer cells treated with aspirin, and also, the suitable internal reference genes were screened by using a microarray and were further identified by using the geNorm and NormFinder softwares, and then were validated in more cell lines and xenografts. We have showed that three traditional internal reference genes, β-actin, GAPDH and α-tubulin, are not suitable for studying gene transcription in colon cancer cells treated with aspirin, and we have identified and validated TMEM208 and PQLC2 as the ideal internal reference genes for detecting the molecular targets of aspirin in colon cancer in vitro and in vivo. This study reveals stable internal reference genes for studying the target genes of aspirin in colon cancer, which will contribute to identify the molecular mechanism behind aspirin preventing colon cancer.

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“…There are multiple reasons for this, one cause may be that ovarian carcinoma is generally detected late, almost 70% of all patients present with advanced stage III and IV cancer, and a number of patients are misdiagnosed (3)(4)(5). The majority of patients suffer from abdominal, gastrointestinal, urinary, or pelvic pain, which rarely lead to timely definitive diagnosis, leading to the generally late detection of ovarian carcinomas (6,7).…”

Section: Introductionmentioning

confidence: 99%

“…The activation of oncogenes and mutation or deletion of tumor suppressor genes are the leading causes of ovarian carcinoma (7,20,21). Therefore, the study of suppressor genes and apoptosis-related genes in carcinogenesis and tumor progression in ovarian carcinoma has drawn increasing attention.…”

Section: Introductionmentioning

confidence: 99%

Downregulation of SASH1 correlates with tumor progression and poor prognosis in ovarian carcinoma

et al. 2016

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Abstract. SAM-and SH3-domain containing 1 (SASH1) is a recently identified tumor suppressor gene that is required in the tumorigenesis of breast and other solid carcinomas. The SASH1 protein contains SH3 and SAM domains, indicating that it may serve an important role in intracellular signal transduction. The purpose of the present study was to investigate the expression of SASH1 in ovarian carcinoma and the correlation between its expression with clinical pathological features and clinical significance, and the effect of SASH1 on cell proliferation, apoptosis and migration of ovarian SKOV3 cells. The human ovarian carcinoma tissues and adjacent normal tissues were collected following surgery. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to detect the expression levels of SASH1 mRNA and protein, respectively. The expression levels of SASH1 mRNA and protein in ovarian carcinoma tissues were significantly lower than that observed in adjacent normal tissues (P<0.05). The expression levels of SASH1 in samples from patients without lymph nodes metastasis and patients with early FIGO stage was lower than those with lymph nodes metastasis and patients with advanced FIGO stage (P<0.05). Flow cytometry analysis and Transwell invasion chamber experiments were used to investigate the effect of SASH1 on the cell proliferation, apoptosis and migration of SKOV3 cells. The recombinant plasmid pcDNA3.1-SASH1 was constructed and transfected into SKOV3 cells. In addition, the SKOV3 cells in the pcDNA3.1-SASH1 group exhibited significantly reduced cell growth, proliferation, and migration ability compared to the empty vector group and normal group (P<0.01). There were a greater number of apoptotic cells in the pcDNA3.1-SASH1 group compared to the empty vector group and normal group (P<0.01). Taken together, these results indicated that SASH1 may be a tumor suppressor gene in ovarian carcinoma, and SASH1 expression inhibited growth, proliferation and migration, and enhanced apoptosis of SKOV3 cells.

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RPL13A as a reference gene for normalizing mRNA transcription of ovarian cancer cells with paclitaxel and 10-hydroxycamptothecin treatments

Cited by 27 publications

References 26 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

Identification of TMEM208 and PQLC2 as reference genes for normalizing mRNA expression in colorectal cancer treated with aspirin

Downregulation of SASH1 correlates with tumor progression and poor prognosis in ovarian carcinoma

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