title: Effect of biological matrix and sample preparation on qPCR quantitation of siRNA drugs in animal tissues.
abstract: BACKGROUND
  Quantitative pharmacokinetic measurement of short nucleotide sequences in animal tissues is critical to the successful development of siRNA-based drugs. Stem-loop qRT-PCR is a sensitive and precise methodology, but the effect of biological matrix and purity of the input sample has yet to be investigated.
  
  
  RESULTS
  The impact of lipid encapsulation, siRNA chemical modification and purity of the biological matrix on the stem-loop qRT-PCR assay was investigated. A comparison of siRNA standard curves in mouse liver homogenates before and after isolation of total RNA uncovered the potential for erroneous measurement due to significant loss of siRNA on purification columns. Recovery of chemically stabilized siRNA was improved by omission of the DNAse I digestion during RNA isolation. The stem-loop qRT-PCR method demonstrated excellent sensitivity and efficiency in mouse liver homogenates, plasma and whole blood. An optimized protocol based on these findings was used to quantitate siRNA in tissues after dosing mice with two different lipid nanoparticle formulations containing siRNA payloads.
  
  
  CONCLUSIONS
  Assay of crude homogenates, whole blood or plasma is more accurate, less resource intensive and more amenable to clinical translation than measurement of column-purified total RNA.
authors.affiliation: Department of RNA Therapeutics, Merck and Co., Inc., West Point, PA 19486, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Seitzer
authors.forename: Hangchun
authors.initials: H
authors.lastname: Zhang
authors.forename: Martin
authors.initials: M
authors.lastname: Koser
authors.forename: Yi
authors.initials: Y
authors.lastname: Pei
authors.forename: Marc
authors.initials: M
authors.lastname: Abrams
mesh_terms: D000818:Animals; D002626:Chemistry, Pharmaceutical; D005069:Evaluation Studies as Topic; D005260:Female; D008081:Liposomes / Q000008:administration & dosage / Q000737:chemistry; D051379:Mice; D053758:Nanoparticles / Q000008:administration & dosage / Q000737:chemistry; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000737:chemistry / Q000302:isolation & purification* / Q000493:pharmacokinetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D008081:Liposomes; D034741:RNA, Small Interfering