Novel Isothermal, Linear Nucleic Acid Amplification Systems for Highly Multiplexed Applications

Kurn, Nurith; Chen, Pengchin; Heath, Joe Don; Kopf-Sill, Anne; Stephens, K; Wang, Shenglong

doi:10.1373/clinchem.2005.053694

Cited by 187 publications

(137 citation statements)

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“…For whole population analyses, >10,000 pooled cells were collected from each group. We used a linear RNA amplification system for the whole transcriptome sequencing (19). The use of such a system prevents reproduction of an error introduced in earlier amplification cycles, a concern in exponential amplification systems.…”

Section: Generation Of a Paclitaxel Tolerance Paradigm In Metastatic mentioning

confidence: 99%

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing

Lee

López-Díaz

Khan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

171

150

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The acute cellular response to stress generates a subpopulation of reversibly stress-tolerant cells under conditions that are lethal to the majority of the population. Stress tolerance is attributed to heterogeneity of gene expression within the population to ensure survival of a minority. We performed whole transcriptome sequencing analyses of metastatic human breast cancer cells subjected to the chemotherapeutic agent paclitaxel at the single-cell and population levels. Here we show that specific transcriptional programs are enacted within untreated, stressed, and drugtolerant cell groups while generating high heterogeneity between single cells within and between groups. We further demonstrate that drug-tolerant cells contain specific RNA variants residing in genes involved in microtubule organization and stabilization, as well as cell adhesion and cell surface signaling. In addition, the gene expression profile of drug-tolerant cells is similar to that of untreated cells within a few doublings. Thus, single-cell analyses reveal the dynamics of the stress response in terms of cell-specific RNA variants driving heterogeneity, the survival of a minority population through generation of specific RNA variants, and the efficient reconversion of stress-tolerant cells back to normalcy.

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Section: Generation Of a Paclitaxel Tolerance Paradigm In Metastatic mentioning

confidence: 99%

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing

Lee

López-Díaz

Khan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

171

150

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“…VHL was quantified using the Taqman VHL primer set (Applied Biosystem, Foster City, CA), using Cyclophillin (Applied Biosystem) as an endogenous control. The relative quantitation of both HIF-1α and VHL was determined by the comparative CT (thermal cycle) method [21].…”

Section: Quantitative Real-time Pcrmentioning

confidence: 99%

Over-expression of hypoxia-inducible factor 1 alpha in ovarian clear cell carcinoma

Lee

Garner

Welch

et al. 2007

Gynecologic Oncology

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Objective-Unlike other histological types of epithelial ovarian carcinoma, ovarian clear cell carcinoma is known to have very poor response to therapy even when discovered in its early stages. Since tumor hypoxia has been shown to be strongly associated with poor prognosis, deregulation of the representative factor of tissue hypoxia; hypoxia-inducible factor 1 alpha (HIF-1α) and related protein; Von Hippel-Lindau (VHL) may be associated with poor prognosis of ovarian clear cell carcinoma.Methods-Immunolocalization of both HIF-1α and VHL was performed on 56 cases of paraffinembedded tissue sections of four different histological types of epithelial ovarian carcinoma and 5 cases of benign ovarian tumors as a control. Quantitative RT-PCR analysis of both HIF1A and VHL was performed on RNA isolated from 61 micro dissected frozen tissues of four different histological types of epithelial ovarian carcinoma and 6 cases of normal ovarian epithelial cells. Expression levels of HIF-1α and VHL in different histological types and correlation between HIF-1α and VHL were determined by nonparametric analysis by Kruskal-Wallis and Spearman's test.Results-HIF-1α expression levels were significantly higher in ovarian clear cell carcinoma than in other histological types (p=0.001). We found no correlation between mRNA and protein expression level in any type of carcinoma specimens. Among endometrioid, serous, and mucinous carcinoma, there were no differences in HIF-1α expression (p=0.643). There was a negative correlation between HIF-1α and VHL in serous (r = −0.661, p=0.027) and in endometrioid carcinoma (r = −0.657 p=0.039), but no correlation was found between HIF-1α and VHL expression levels in ovarian clear cell carcinoma (p=0.60). Conclusions-The results suggest that the role of hypoxia may change according to the histological type of ovarian carcinoma. High expression of HIF-1α and its independence from VHL in ovarian clear cell carcinoma may confer chemoresistance in this histological type.

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“…The single primer isothermal amplification (SPIA) was developed by NuGen TM in 2005, which provide various SPIA products [192]. This reaction uses a chimeric primer consisting of DNA at the 3´-end and RNA at the 5´-end.…”

Section: Various Other Methodsmentioning

confidence: 99%

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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Novel Isothermal, Linear Nucleic Acid Amplification Systems for Highly Multiplexed Applications

Cited by 187 publications

References 13 publications

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing

Single-cell analyses of transcriptional heterogeneity during drug tolerance transition in cancer cells by RNA sequencing

Over-expression of hypoxia-inducible factor 1 alpha in ovarian clear cell carcinoma

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

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