Quantification of dsRNA using stable isotope labeling dilution liquid chromatography/mass spectrometry

<jats:sec><jats:title>Rationale</jats:title><jats:p>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.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Stable isotope labeled dsRNA was synthesised both <jats:italic>in vivo</jats:italic> (<jats:sup>15</jats:sup>N) and <jats:italic>in vitro</jats:italic> (<jats:sup>13</jats:sup>C,<jats:sup>15</jats:sup>N‐guanosine‐containing dsRNA) prior to purification and quantification. The stable isotope labeled dsRNA standards were subsequently spiked into total RNA extracted from <jats:styled-content style="fixed-case"><jats:italic>E. coli</jats:italic></jats:styled-content> 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 oligonucleotide pairs.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>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<jats:sup>8</jats:sup> <jats:styled-content style="fixed-case"><jats:italic>E. coli</jats:italic></jats:styled-content> cells expressing 765 bp and 401 bp dsRNAs, respectively.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Stable isotope labeling of dsRNA in conjunction with MS enabled the characterisation and quantification of dsRNA in complex total RNA mixtures.</jats:p></jats:sec>