An mRNA amplification procedure with directional cDNA cloning and strand-specific cRNA synthesis for comprehensive gene expression analysis

Ohtsuka, Satoko; Iwase, Katsuro; Kato, Masaki; Seki, Naohiko; Shimizu-Yabe, Atsuko; Miyauchi, Osamu; Sakao, Eiko; Kanazawa, Masato; Yamamoto, Shozo; Kohno, Yoichi; Takao, Masaki

doi:10.1016/j.ygeno.2004.06.012

Cited by 12 publications

(14 citation statements)

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“…Techniques based on the large scale measurement of mRNA expression can be easily applied for evaluating the complexity and influence of gene expression on cellular metabolism and processes leading to neoplastic transformation (22,23). Continual advancement of such techniques, along with rigorous analysis, is the key to identifying the most relevant genetic changes and correlating them with patient outcome.…”

Section: Introductionmentioning

confidence: 99%

Expression of genes encoding extracellular matrix proteins: A macroarray study

et al. 2014

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Endometrial cancer (EC) is one of the most common gynecological malignancies in Poland, with well-established risk factors. Genetic instability and molecular alterations responsible for endometrial carcinogenesis have been systematically investigated. The aim of the present study was to investigate, by means of cDNA macroarrays, the expression profiles of genes encoding extracellular matrix (ECM) proteins in ECs. Tissue specimens were collected during surgical procedures from 40 patients with EC, and control tissue was collected from 9 patients with uterine leiomyomas. RNA was isolated and RT-PCR with radioisotope-labeled cDNA was performed. The levels of ECM protein gene expression in normal endometrial tissues were compared to the expression of these genes in EC specimens. Statistically significant differences in gene expression, stratified by clinical stage of the ECs, were detected for aggrecan, vitronectin, tenascin R, nidogen and two collagen proteins: type VIII chain α1 and type XI chain α2. All of these proteins were overexpressed in stage III endometrial carcinomas compared to levels in stage I and II uterine neoplasms. In conclusion, increased expression of genes encoding ECM proteins may play an important role in facilitating accelerated disease progression of human ECs.

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Section: Introductionmentioning

confidence: 99%

Expression of genes encoding extracellular matrix proteins: A macroarray study

et al. 2014

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“…Evolutions of the PCR-based approach have addressed many of these concerns. Some groups have opted to combine both PCR and T7/IVT approaches, which helps balance out some of the problems of both techniques [37][38][39].…”

Section: Amplification Approachesmentioning

confidence: 99%

RNA Amplification Strategies: Toward Single‐Cell Sensitivity

Stickle¹,

Iscove²,

Virtanen³

et al. 2010

Genomics

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“…PCR-based cDNA amplification can be categorized as template switching (TS)-PCR [52,68,69], random PCR [70] and 3' tailing with 5' adaptor ligation PCR [71] based on the generation of a 5' anchor sequence which provides a platform for 5' primer annealing. TS-PCR employs the same template switch mechanism in ds-cDNA generation and in the amplification of ds-cDNA using 5' TS primer II (truncated TS primer) and 3' oligo dT or dT-T7 primers (depending upon the primer used in the first strand cDNA synthesis).…”

Section: Introductionmentioning

confidence: 99%

“…Ds-cDNA can then be amplified under one oligo dT primer or dT-adaptor primer if an adaptor sequence is attached [66]. Direct adaptor ligation is another alternative way to generate ds-cDNA with a known anchor sequence at the 5' end [71]. In this way, single strand cDNA is generated using oligo dT primers immobilized onto magnetic beads and second strand cDNA is completed by Van Gelder and Eberwine's ds-cDNA generation method.…”

Section: Introductionmentioning

confidence: 99%

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RNA amplification for successful gene profiling analysis

Wang

2005

J Transl Med

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Gene profilingcDNA microarrayRNA amplificationpolymerasehigh throughput analysis. AbstractThe study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene-specific, unbiased transcriptome wide amplification accurately maintains proportionality among all RNA species within a given specimen. This allows the utilization of clinical material obtained with minimally invasive methods such as fine needle aspirates (FNA) or cytological washings for high throughput functional genomics studies. This review provides a comprehensive and updated discussion of the literature in the subject and critically discusses the main approaches, the pitfalls and provides practical suggestions for successful unbiased amplification of the whole transcriptome in clinical samples.

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An mRNA amplification procedure with directional cDNA cloning and strand-specific cRNA synthesis for comprehensive gene expression analysis

Cited by 12 publications

References 63 publications

Expression of genes encoding extracellular matrix proteins: A macroarray study

Expression of genes encoding extracellular matrix proteins: A macroarray study

RNA Amplification Strategies: Toward Single‐Cell Sensitivity

RNA amplification for successful gene profiling analysis

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