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Anal Biochem. 2009 Feb 15;385(2):386-8. doi: 10.1016/j.ab.2008.11.037. Epub 2008 Dec 6.

Quantitation of full-size small interfering RNA by tailing with terminal deoxynucleotidyl transferase and reverse transcription-polymerase chain reaction analysis.

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GeneCare Research Institute, Kamakura, Kanagawa, Japan.


Accurate estimation of small interfering RNA (siRNA) concentration in cells and blood is increasingly important for pharmacokinetic studies required to develop siRNA drugs. We report a method that detects siRNA having 3'-terminal deoxynucleotide overhangs, such as 3'-dTdT, present in most chemically synthesized siRNAs. Short overhangs were elongated to oligo-dG by incubation with terminal deoxynucleotidyl transferase and dGTP and were used as priming sites for reverse transcription of siRNA to complementary DNA (cDNA). The resultant cDNA was used as a template for quantitation by polymerase chain reaction. This method was reliable for determining the pharmacokinetics of siRNA in blood of injected mice.

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