INTRODUCTION: 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.
INTRODUCTION: 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: Bin Shi; Ed Keough; Andrea Matter; Karen Leander; Stephanie Young; Ed Carlini; Alan B Sachs; Weikang Tao; Marc Abrams; Bonnie Howell; Laura Sepp-Lorenzino Journal: J Histochem Cytochem Date: 2011-08 Impact factor: 2.479