Developing an Analytical Method for Separating and Quantifying RNA Generated in In Vitro Transcription Reactions

The generation of run-off transcripts from in vitro transcription reactions is a useful technique in the study of transcription regulation. There are currently limited ways in which these run-off transcripts can be analyzed, and few that are truly quantitative. Our aim is to establish a method using ion-pair reverse phase high performance liquid chromatography (IP RP HPLC) to analyze RNA transcripts from in vitro transcription reactions. We were able to demonstrate that we could recapitulate the separation of DNA based on size using our IP RP HPLC method. The application of this method was also able to effectively separate RNA based on size in the size range of 281 – 1908 bp. Using a simple in vitro transcription system with T7 RNA polymerase and a linear DNA template with a single promoter, we showed detection of the RNA run-off transcript in the size range demonstrated. We would like to apply this method to more complex in vitro transcription systems and demonstrate the ability to quantify RNA using IP RP HPLC

Quantification of DNA Products Using Ion-Pair Reverse Phase Liquid Chromatography

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. Quantitative analysis of the DNA templates can be done via ion-pair reverse-phase high performance liquid chromatography (IP-RP HPLC), a high-resolution technique in separating DNA based on size. Using IP-RP HPLC our aim is to assess the lower limits of separation, and our quantification method is based on measuring peak area and the peak height

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

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