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Nucleic Acid Ther. 2016 Apr;26(2):86-92. doi: 10.1089/nat.2015.0578. Epub 2015 Nov 23.

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo.

Author information

1
1 RNA Therapeutics Institute, University of Massachusetts Medical School , Worcester, Massachusetts.
2
2 Department of Molecular Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.
3
3 Department of Medicine, University of Massachusetts Medical School , Worcester, Massachusetts.

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.

PMID:
26595721
PMCID:
PMC4834514
DOI:
10.1089/nat.2015.0578
[Indexed for MEDLINE]
Free PMC Article

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