Kung, A-W., Kilby, P.M., Portwood, D.E. et al. (1 more author) (2018) Quantification of dsRNA using stable isotope labeling dilution liquid chromatography mass spectrometry. Rapid Communications in Mass Spectrometry, 32 (7). pp. 590-596. ISSN 0951-4198
Abstract
RATIONALE: Recent developments in RNA interference (RNAi) have created a need for cost-effective and large scale synthesis of double stranded RNA (dsRNA), in conjunction with high throughput analytical techniques to fully characterise and accurately quantify dsRNA prior to downstream RNAi applications. METHODS: Stable isotope labeled dsRNA was synthesised both in vivo (15N) and in vitro (13C,15N guanosine-containing dsRNA) prior to purification and quantification. The stable isotope labeled dsRNA standards were subsequently spiked into total RNA extracted from E. coli engineered to express dsRNA. RNase mass mapping approaches were subsequently performed using LC-ESI-MS for both the identification and absolute quantification of the dsRNA using the ratios of the light and heavy oligonucleotides pairs. RESULTS: Absolute quantification was performed based on the resulting light and heavy oligoribonicleotides identified using mass spectrometry. Using this approach we determined that 624.6 ng/μl and 466.5 ng/μl of dsRNA was present in 80 μl total RNA extracted from 108E. coli cells expressing 765 bp and 401 bp dsRNA respectively. CONCLUSIONS: Stable isotopic labelling of dsRNA in conjunction with mass spectrometry enabled the characterisation and quantification of dsRNA in complex total RNA mixtures.
Metadata
Item Type: | Article |
---|---|
Authors/Creators: |
|
Copyright, Publisher and Additional Information: | © 2018 Wiley. This is an author produced version of a paper subsequently published in Rapid Communications in Mass Spectrometry. Uploaded in accordance with the publisher's self-archiving policy. |
Keywords: | RNA quantification; RNase mass mapping; dsRNA; mass spectrometry |
Dates: |
|
Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Funding Information: | Funder Grant number ENGINEERING AND PHYSICAL SCIENCE RESEARCH COUNCIL (EPSRC) EP/D033713/1 SYNGENTA AG PO: 8700549715 BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL (BBSRC) BB/M012166/1 SYNGENTA LTD PO: 8700549715 ENGINEERING AND PHYSICAL SCIENCE RESEARCH COUNCIL (EPSRC) None |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 20 Feb 2018 13:15 |
Last Modified: | 09 Sep 2020 10:37 |
Status: | Published |
Publisher: | Wiley |
Refereed: | Yes |
Identification Number: | 10.1002/rcm.8074 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:127671 |