Details

Autor(en) / Beteiligte

• Coles, Andrew H
• Osborn, Maire F
• Alterman, Julia F
• Turanov, Anton A
• Godinho, Bruno M D C
• Kennington, Lori
• Chase, Kathryn
• Aronin, Neil
• Khvorova, Anastasia

Titel

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

Ist Teil von

• Nucleic acid therapeutics, 2016-04, Vol.26 (2), p.86

Ort / Verlag

United States

Erscheinungsjahr

2016

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.