Abstract 1307: Assessment of long IVT mRNA fragments with the Fragment Analyzer system

Abstract

Quality control analysis of long in vitro transcribed (IVT) RNAs is challenging due to the lack of commercially available kits for sizing RNA over 6,000 nt, thus, to analyze long IVT RNAs, an extended separation method was developed for the Agilent 5200 Fragment Analyzer using a commercially available RNA ladder. IVT mRNA was produced using the RiboMAX Large Scale RNA Production System (Promega). RNA transcripts 9,000 and 10,000 nt in length were diluted in nuclease-free water and separated on the Agilent 5200 Fragment Analyzer system with the Agilent RNA kit (15 nt) (p/n DNF-471) with the following modifications. The Lonza RNA marker (Lonza) (long RNA Ladder) replaced the Agilent RNA Ladder and was diluted in nuclease-free water to 96 ng/μL. The long RNA Ladder was added to the Agilent RNA Diluent Marker (15 nt) (p/n DNF-369-0004) according to the RNA kit protocol. The standard separation method (8 kV for 45 minutes) for the RNA kit (15 nt) was manually altered to the extended RNA method (4 kV for 90 minutes) in the Agilent 5200 Fragment Analyzer software for fragments greater than 6,000 nt. The extended RNA method with the long RNA Ladder was employed for all subsequent runs. The long RNA Ladder separated with the extended method displayed enhanced resolution compared to separation with the standard method as seen by the increased spacing between the ladder peaks and the increased sharpness of the 9,000 nt peak. The extended RNA method reported a lower average sizing percent error (-0.7 %) over the entire concentration range of the kit compared to the standard RNA method (8.4 %) for the 9,000 nt sample. The 10,000 nt IVT mRNA average percent sizing error was similar between the two separation methods throughout the dilution series. Using the extended method, the 9,000 and 10,000 nt IVT mRNA samples were successfully analyzed with good sizing precision and accuracy, demonstrating that a commercially available RNA ladder can be used with the Fragment Analyzer system to provide accurate sizing of large IVT mRNA samples. The extended RNA method is recommended when extremely accurate sizing or high resolution is needed for determining the presence of degradation or sizing differences from incomplete transcription in IVT mRNA samples longer than 6,000 nt. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Kyle D. Luttgeharm, Solange Borg, Chava Pocernich. Assessment of long IVT mRNA fragments with the Fragment Analyzer system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1307.