Submission Title

Quantification of In-Vitro Transcription RNA Produced Using Ion-pair Reverse Phase Liquid Chromatography

Location

Jereld R. Nicholson Library: Grand Avenue

Subject Area

Biochemistry

Description

The transcription of DNA via RNA polymerases is a fundamental process in cellular systems. In eukaryotic cells, we observe transcription in the nucleus (via genomic DNA) as well as in the mitochondria (via mitochondrial DNA). There are many tools available to investigate nuclear transcription; however, few tools exist to study mitochondrial transcription even though the mitochondrial DNA encodes several essential proteins. Recently an in vitro transcription system using purified mitochondrial transcription proteins, including the mitochondrial RNA polymerase, and linear mitochondrial DNA templates has been developed. Run-off RNA transcripts of in vitro transcription reactions have traditionally been analyzed using denaturing polyacrylamide gel electrophoresis (PAGE) and detection of labeled-UTP incorporated into the transcripts. Although this technique is sufficient for the detection of multiple run-off products, quantitative analysis of RNA transcripts by denaturing PAGE is difficult. Ion-pair reverse-phase high performance liquid chromatography (IP-RP HPLC) has long been used as a high-resolution technique in separating DNA based on the number of base pairs. Using denaturing conditions, we have successfully separated and quantified DNA run-off transcript samples of mixed sizes. Our quantification method is based on measuring peak area, height of the peak, retention time, and the use of internal standards to determine transcript size. This method of quantitatively detecting run-off DNA can be applied to the analysis of the mitochondrial in vitro transcription assay due to similar properties and characteristics.

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Quantification of In-Vitro Transcription RNA Produced Using Ion-pair Reverse Phase Liquid Chromatography

Jereld R. Nicholson Library: Grand Avenue

The transcription of DNA via RNA polymerases is a fundamental process in cellular systems. In eukaryotic cells, we observe transcription in the nucleus (via genomic DNA) as well as in the mitochondria (via mitochondrial DNA). There are many tools available to investigate nuclear transcription; however, few tools exist to study mitochondrial transcription even though the mitochondrial DNA encodes several essential proteins. Recently an in vitro transcription system using purified mitochondrial transcription proteins, including the mitochondrial RNA polymerase, and linear mitochondrial DNA templates has been developed. Run-off RNA transcripts of in vitro transcription reactions have traditionally been analyzed using denaturing polyacrylamide gel electrophoresis (PAGE) and detection of labeled-UTP incorporated into the transcripts. Although this technique is sufficient for the detection of multiple run-off products, quantitative analysis of RNA transcripts by denaturing PAGE is difficult. Ion-pair reverse-phase high performance liquid chromatography (IP-RP HPLC) has long been used as a high-resolution technique in separating DNA based on the number of base pairs. Using denaturing conditions, we have successfully separated and quantified DNA run-off transcript samples of mixed sizes. Our quantification method is based on measuring peak area, height of the peak, retention time, and the use of internal standards to determine transcript size. This method of quantitatively detecting run-off DNA can be applied to the analysis of the mitochondrial in vitro transcription assay due to similar properties and characteristics.