Quantification of dsRNA using stable isotope labeling dilution
liquid chromatography/mass spectrometry
Peter M. Kilby
David E. Portwood
Mark J. Dickman
Department of Chemical and Biological
Engineering, Mappin Street, University of
Sheffield, Sheffield S1 3JD, UK
Syngenta, Jealott's Hill International
Research Centre, Bracknell, Berkshire RG42
M.J. Dickman, Department of Chemical and
Biological Engineering, Mappin Street,
University of Sheffield, Sheffield S1 3JD, UK.
Biotechnology and Biological Sciences
Research Council, Grant/Award Number:
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.
Stable isotope labeled dsRNA was synthesised both in vivo (
N) and in vitro
N‐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 liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI‐MS) for both
the identification and absolute quantification of the dsRNA using the ratios of the light and heavy
Absolute quantification was performed based on the resulting light and heavy
oligoribonucleotides identified using MS. 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 10
E. coli cells
expressing 765 bp and 401 bp dsRNAs, respectively.
Stable isotope labeling of dsRNA in conjunction with MS enabled the
characterisation and quantification of dsRNA in complex total RNA mixtures.
Exploitation of the RNA interference (RNAi) pathway to block the
expression of specific genes holds considerable promise for the
development of novel RNAi‐based insect management strategies.
There are a wide range of future potential applications of RNAi to
control agricultural insect pests as well as its use for prevention of
diseases in beneficial insects. Recent developments in RNAi have
created a need for cost‐effective, large‐scale synthesis of double‐
stranded RNA (dsRNA), which in turn requires robust analytical
techniques to fully characterise and accurately quantify dsRNA prior to
RNAi applications. A wide range of dsRNA products can be generated
either via bacterial expression systems, in planta or in vitro transcription.
The development of suitable analytical methods to characterise the
dsRNA products remains a significant challenge.
E. coli‐mediated delivery of dsRNA has been reported in
Furthermore, a number of RNAi‐based insect management
strategies have also employed the ingestion of bacteria‐expressing
application of chemically synthesised dsRNA
plants expressing dsRNA.
To ensure RNAi gene silencing using the
above approaches, it is important to both produce and deliver the
required amounts of dsRNA. Therefore, the necessary analytical tools
to quantify the dsRNA are important to both optimise production
strategies and ensure delivery of the required amounts of dsRNA.
Mass spectrometry (MS) is a powerful approach for the analysis
and direct characterisation of nucleic acids. RNase mass mapping
methods have been performed to identify and characterise a wide
range of RNAs.
Prior to MS analysis, purification of the RNA of
interest using high‐performance liquid chromatography (HPLC) is an
essential step. For further LC/MS analysis, specific RNase digestions
are performed in order to produce smaller oligoribonucleotide fragments,
which are then amenable for direct on‐line LC separation and MS
analysis. RNase mass mapping methods have been widely employed for
the identification of RNA and RNA post‐transcriptional modifications.
In addition, we have recently developed RNase mass mapping
approaches to identify and characterise dsRNA.
Recent MS studies have focused on the development of more
quantitative approaches by using isotopic labeling in conjunction with
We were the first to introduce the use of
Received: 29 September 2017 Revised: 26 January 2018 Accepted: 26 January 2018
590 Copyright © 2018 John Wiley & Sons, Ltd. Rapid Commun Mass Spectrom. 2018;32:590–596.wileyonlinelibrary.com/journal/rcm