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

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.