Quantitation of full-size small interfering RNA by tailing with terminal deoxynucleotidyl transferase and reverse transcription-polymerase chain reaction analysis.
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.
Futami
Kazunobu
K
GeneCare Research Institute, Kamakura, Kanagawa, Japan.
Furuichi
Yasuhiro
Y
D000818:Animals; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D018076:DNA, Complementary; D003848:Deoxyguanine Nucleotides; D051379:Mice; D034741:RNA, Small Interfering / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics; D012107:Research Design; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D018076:DNA, Complementary; D003848:Deoxyguanine Nucleotides; D034741:RNA, Small Interfering; C029603:deoxyguanosine triphosphate; D004253:DNA Nucleotidylexotransferase