Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and multicolor detection

Odin, Elisabeth; Wettergren, Yvonne; Larsson, Lars; Larsson, Per‐Göran; Gustavsson, Bengt

doi:10.1016/s0378-4347(99)00333-3

Cited by 7 publications

(3 citation statements)

References 9 publications

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“…Several studies have also used CE-LIF to analyze RT-PCR products of individual genes [3][4][5] or to qualitatively detect multiple PCR products [13,14]. The method described herein is the first report of direct analysis of mRNA or rRNA in which not only the quantity of RNA species can be detected but also the size of the RNA.…”

Section: Discussionmentioning

confidence: 99%

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Goldsmith¹,

Ntuen²,

Goldsmith³

2007

Analytical Biochemistry

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Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and is often accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. While numerous methods are commonly used to monitor gene expression, including northern blotting, real time polymerase chain reaction and RNase protection, each has its own drawbacks and limitations. Capillary electrophoresis with laser induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something which is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of Discoidin Domain Receptor 2 mRNA in cardiac tissue.

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Section: Discussionmentioning

confidence: 99%

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Goldsmith¹,

Ntuen²,

Goldsmith³

2007

Analytical Biochemistry

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“…Integration of PCR and CE for multiplexed assays was originally suggested as end-point PCR amplification followed by analysis and quantification of the amplified targets by CE in which each target DNA was identified based on amplicon length. 26 This approach was successfully applied to gene expression analysis 22 , 27 and diagnostic applications 28 , 29 , 30 , 31 using end-point PCR detection. However, real-time PCR is preferred to endpoint PCR due to better precision and broader dynamic range of quantitative measurements.…”

Section: Discussionmentioning

confidence: 99%

Scalable Transcriptional Analysis Routine—Multiplexed Quantitative Real-Time Polymerase Chain Reaction Platform for Gene Expression Analysis and Molecular Diagnostics

Garcia

Dowding

Stanton

et al. 2005

The Journal of Molecular Diagnostics

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We report the development of a new technology for simultaneous quantitative detection of multiple targets in a single sample. Scalable transcriptional analysis routine (STAR) represents a novel integration of reverse transcriptase-polymerase chain reaction and capillary electrophoresis that allows detection of dozens of gene transcripts in a multiplexed format using amplicon size as an identifier for each target. STAR demonstrated similar or better sensitivity and precision compared to two commonly used methods, SYBR Green-based and TaqMan probe-based real-time reverse transcriptase-polymerase chain reaction. STAR can be used as a flexible platform for building a variety of applications to monitor gene expression, from single gene assays to assays analyzing the expression level of multiple genes. Using severe acute respiratory syndrome (SARS) corona virus as a model system, STAR technology detected single copies of the viral genome in a two-gene multiplex. Blinded studies using RNA extracted from various tissues of a SARS-infected individual showed that STAR correctly identified all samples containing SARS virus and yielded negative results for non-SARS control samples. Using alternate priming strategies, STAR technology can be adapted to transcriptional profiling studies without requiring a priori sequence information. Thus, STAR technology offers a flexible platform for development of highly multiplexed assays in gene expression analysis and molecular diagnostics.

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“…A widely used strategy for the preparation of fragments from coding regions of genomic DNA utilizes reverse transcription of mRNA to cDNA and its subsequent PCR amplification (RT-PCR). ,− Another method used in cancer diagnostics is the monitoring of telomerase activity, a useful biomarker for early detection of cancer. Telomerase activity correlates with tumor progression, indicating that tumors expressing this enzyme exhibit aggressive clinical behavior and unlimited proliferation and immortalization of cells. ,− Many other useful applications of CE in cancer research ,,− and clinical diagnostics ,,,,,− have been published including the implementation of microfluidic devices. ,,,, …”

Section: 7 Cancermentioning

confidence: 99%

DNA Diagnostics by Capillary Electrophoresis

Klepárnı́k

Boček

2007

Chem. Rev.

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Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and multicolor detection

Cited by 7 publications

References 9 publications

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Scalable Transcriptional Analysis Routine—Multiplexed Quantitative Real-Time Polymerase Chain Reaction Platform for Gene Expression Analysis and Molecular Diagnostics

DNA Diagnostics by Capillary Electrophoresis

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