title: A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo.
abstract: Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene(®) branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo.
authors.affiliation: 1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.|2 Department of Molecular Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Coles
authors.affiliation: 1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.|2 Department of Molecular Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
authors.forename: Maire F
authors.initials: MF
authors.lastname: Osborn
authors.affiliation: 1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.|2 Department of Molecular Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
authors.forename: Julia F
authors.initials: JF
authors.lastname: Alterman
authors.affiliation: 1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.|2 Department of Molecular Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
authors.forename: Anton A
authors.initials: AA
authors.lastname: Turanov
authors.affiliation: 1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.|2 Department of Molecular Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
authors.forename: Bruno M D C
authors.initials: BM
authors.lastname: Godinho
authors.affiliation: 1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.|3 Department of Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
authors.forename: Lori
authors.initials: L
authors.lastname: Kennington
authors.affiliation: 1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.|3 Department of Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Chase
authors.affiliation: 1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.|3 Department of Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
authors.forename: Neil
authors.initials: N
authors.lastname: Aronin
authors.affiliation: 1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.|2 Department of Molecular Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Khvorova
mesh_terms: D000818:Animals; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D057166:High-Throughput Screening Assays; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000627:therapeutic use; D015203:Reproducibility of Results
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering