Comparison of different standards for real-time PCR-based absolute quantification

Dhanasekaran, Senthil; Doherty, T. Mark; Kenneth, John

doi:10.1016/j.jim.2010.01.004

Cited by 189 publications

(133 citation statements)

References 16 publications

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“…Reliable standards are essential in qPCR analysis. Therefore, we selected a cloned circular plasmid, which is more stable than PCR products (20). The four miRNAs (miR-21, miR-31, miR-96 and miR-135b) and U6 snRNA from the HT29 cells were reversetranscribed.…”

Section: Methodsmentioning

confidence: 99%

Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer

et al. 2012

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Abstract. The aim of this study was to determine the expression of miR-21, miR-31, miR-96 and miR-135b in 52 paired colorectal cancer (CRC) tissues and to analyze the correlation between microRNAs (miRNAs) and clinicopathological features. We developed a quantification method that relies on a standard plot, constructed from known concentrations of standards, in order to measure the number of miRNAs. In addition to this, we analyzed the expression levels of miR-21, miR-31, miR-96 and miR-135b in 52 cases of primary CRC and corresponding normal mucosal tissue using real-time PCR with SYBR-Green I. An independent sample t-test was used to compare the differential expression between tumor tissues and normal mucosal tissues. The Mann-Whitney U and Kruskall-Wallis tests were used to compare the correlation between miRNA expression levels and clinicopathological features. The expression of miR-21, miR-31, miR-96 and miR-135b was upregulated in the CRC tissues compared to normal mucosal tissues (P<0.05). Furthermore, miR-21 and miR-135b were positively correlated with the clinical stage (P=0.048 and P=0.029, respectively), while miR-96 and miR-135b were correlated with liver metastasis (P=0.006 and P=0.013, respectively). Our results suggest that miR-21, miR-31, miR-96 and miR-135b may function in the process of CRC development and progression. miR-135b levels in particular may correlate with the degree of malignancy. IntroductionColorectal cancer (CRC) is the third most common type of malignant neoplasm and the third leading cause of cancer-related mortalities worldwide (1). Conventional pathological diagnosis is traumatic, with the majority of cases being detected in the later stage. Early detection of CRC has puzzled clinicians and scientists for years. Even with annual fecal occult-blood testing, which has decreased the 13-year cumulative mortality rate from CRC by 33% (2), the outcome remains poor in patients with advanced disease. Only CRC diagnosed at an early stage is likely to be cured by surgical resection. Genetic alterations present in CRC, including those in APC, K-ras or p53, do not demonstrate a confirmed correlation between their mutation rate and clinical stage (3). Therefore, a reliable, sensitive and specific molecular diagnostic test for CRC is highly desirable from a clinical perspective.Accumulating evidence suggests that microRNAs (miRNAs) play a crucial role in the tumorigenesis and prognosis of cancer (4-7). miRNAs are non-coding, single-stranded RNAs of 18-25 nucleotides in length, which are able to regulate gene expression by inhibiting translation or decreasing stability of target mRNAs. Over the past few years, interest in the identification, detection and utilization of miRNA molecules has expanded rapidly. Bioinformatic analysis suggests that up to 30% of human genes may be regulated by miRNAs, despite the fact that they only constitute approximately 1% of the human genome (8,9).Changes in miRNA expression have been observed in a variety of human tumors, including breast (5), prostate (6),...

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Section: Methodsmentioning

confidence: 99%

Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer

et al. 2012

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“…Reaction mixtures and PCR conditions are described in Epelde et al (2014). Standards were made from plasmids containing the target sequence (Dhanasekaran et al, 2010).…”

Section: Presence Of Contaminantsmentioning

confidence: 99%

Characterization of Composted Organic Amendments for Agricultural Use

Epelde

Jauregi

Urra

et al. 2018

Front. Sustain. Food Syst.

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“…Real-time polymerase chain reaction analysis was conducted using TaqMan primer probe sets with quantification relative to a standard curve of the gene copy number (28).…”

Section: Quantitative Real-time Polymerase Chain Reactionmentioning

confidence: 99%

Regulation and Function of Epithelial Secreted Phospholipase A₂ Group X in Asthma

Hallstrand

Lü

Altemeier

et al. 2013

Am J Respir Crit Care Med

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Rationale: Indirect airway hyperresponsiveness (AHR) is a fundamental feature of asthma that is manifest as exercise-induced bronchoconstriction (EIB). Secreted phospholipase A 2 group X (sPLA 2 -X) plays a key role in regulating eicosanoid formation and the development of inflammation and AHR in murine models. Objectives: We sought to examine sPLA 2 -X in the airway epithelium and airway wall of patients with asthma, the relationship to AHR in humans, and the regulation and function of sPLA 2 -X within the epithelium. Methods: We precisely phenotyped 34 patients with asthma (19 with and 15 without EIB) and 10 normal control subjects to examine in vivo differences in epithelial gene expression, quantitative morphometry of endobronchial biopsies, and levels of secreted protein. The regulation of sPLA 2 -X gene (PLA2G10) expression was examined in primary airway epithelial cell cultures. The function of epithelial sPLA 2 -X in eicosanoid formation was examined using PLA 2 inhibitors and murine tracheal epithelial cells with Pla2g10 deletion. Measurements and Main Results: We found that sPLA 2 -X protein is increased in the airways of patients with asthma and that epithelial-derived sPLA 2 -X may be increased in association with indirect AHR. The expression of sPLA 2 -X increases during in vitro epithelial differentiation; is regulated by inflammatory signals including tumor necrosis factor, IL-13, and IL-17; and is both secreted from the epithelium and directly participates in the release of arachidonic acid by epithelial cells. Conclusions: These data reveal a relationship between epithelialderived sPLA 2 -X and indirect AHR in asthma and that sPLA 2 -X serves as an epithelial regulator of inflammatory eicosanoid formation. Therapies targeting epithelial sPLA 2 -X may be useful in asthma.Keywords: airway hyperresponsiveness; asthma; eicosanoid; epithelial cell; secretory phospholipase A 2Indirect airway hyperresponsiveness (AHR) refers to the propensity to develop airway narrowing in response to stimuli such as exercise, osmotic challenge, or adenosine that cause airflow obstruction via inflammatory or neuronal cells that release mediators (1). Exercise-induced bronchoconstriction (EIB) is a prototypical feature of indirect AHR in asthma that occurs in about 30 to 50% of subjects with asthma in cross-sectional studies (2, 3). Prior studies have identified epithelial shedding into the airway lumen and increased production of inflammatory eicosanoids such as leukotrienes in the airways of patients with EIB (4-8). The basis for the dysregulation of eicosanoids in asthma is incompletely understood. The rate-limiting step in eicosanoid biosynthesis is the release of unesterified arachidonic acid (AA) from the sn-2 position of membrane phospholipids by phospholipase A 2 (PLA 2 ) (9). Recent work has uncovered 10 mammalian secreted PLA 2 s (sPLA 2 s) that may coordinate eicosanoid synthesis with the well-described cytosolic PLA 2 -a (i.e., cPLA 2 a) (10-12). sPLA 2 s have several other inflammatory functions, including ...

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Comparison of different standards for real-time PCR-based absolute quantification

Cited by 189 publications

References 16 publications

Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer

Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer

Characterization of Composted Organic Amendments for Agricultural Use

Regulation and Function of Epithelial Secreted Phospholipase A₂ Group X in Asthma

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Comparison of different standards for real-time PCR-based absolute quantification

Cited by 189 publications

References 16 publications

Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer

Expression of miR-21, miR-31, miR-96 and miR-135b is correlated with the clinical parameters of colorectal cancer

Characterization of Composted Organic Amendments for Agricultural Use

Regulation and Function of Epithelial Secreted Phospholipase A2 Group X in Asthma

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Regulation and Function of Epithelial Secreted Phospholipase A₂ Group X in Asthma