PMID- 33016168
OWN - NLM
STAT- MEDLINE
DCOM- 20220218
LR  - 20220830
IS  - 2472-5560 (Electronic)
IS  - 2472-5552 (Linking)
VI  - 26
IP  - 2
DP  - 2021 Feb
TI  - An Automated High-Throughput Fluorescence In Situ Hybridization (FISH) Assay 
      Platform for Use in the Identification and Optimization of siRNA-Based 
      Therapeutics.
PG  - 281-291
LID - 10.1177/2472555220960045 [doi]
AB  - Since the revolutionary discovery of RNA interference (RNAi) more than 20 years 
      ago, synthetic small interfering RNAs (siRNAs) have held great promise as 
      therapeutic agents for treating human diseases by the specific knockdown of 
      disease-causing gene products. To facilitate the development of siRNA 
      therapeutics, a robust, high-throughput in vitro assay for measuring gene 
      silencing is imperative during the initial siRNA lead sequence identification 
      and, later, during the lead optimization with chemically modified siRNAs. There 
      are several potential assays for measuring gene expression. Quantitative reverse 
      transcription PCR (qRT-PCR) has been widely used to quantitate messenger RNA 
      (mRNA). This method has a few disadvantages, however, such as the requirement for 
      RNA isolation, complementary DNA (cDNA) generation, and PCR reaction, which are 
      labor-intensive, limit the assay throughput, and introduce variability. We chose 
      a high-content imaging assay, bDNA FISH, that combines the branched DNA (bDNA) 
      technology with fluorescence in situ hybridization (FISH) to measure gene 
      silencing by siRNAs because it is sensitive and robust with a short reagent 
      procurement and assay development time. We also built a fully automated 
      liquid-handling platform for executing bDNA FISH assays to increase throughput, 
      and the system has a capacity of generating 192 concentration-response curves in 
      a single run. We have successfully developed and executed the bDNA FISH assays 
      for multiple targets using this automated platform to identify and optimize siRNA 
      candidate molecules. Examples of the bDNA FISH assay for selected targets are 
      presented.
FAU - Dou, Hui H
AU  - Dou HH
AD  - Discovery Technologies, Amgen Research, South San Francisco, CA, USA.
FAU - Mallari, Rommel
AU  - Mallari R
AD  - Discovery Technologies, Amgen Research, South San Francisco, CA, USA.
FAU - Pipathsouk, Andrew
AU  - Pipathsouk A
AD  - Discovery Technologies, Amgen Research, South San Francisco, CA, USA.
FAU - Das, Amrita
AU  - Das A
AD  - Cardiometabolic Disorders, Amgen Research, South San Francisco, CA, USA.
FAU - Lo, Mei-Chu
AU  - Lo MC
AUID- ORCID: 0000-0003-3766-2094
AD  - Discovery Technologies, Amgen Research, South San Francisco, CA, USA.
LA  - eng
PT  - Journal Article
DEP - 20201005
PL  - United States
TA  - SLAS Discov
JT  - SLAS discovery : advancing life sciences R & D
JID - 101697563
RN  - 0 (RNA, Small Interfering)
SB  - IM
MH  - *Automation
MH  - Drug Discovery/*methods/standards
MH  - Genetic Therapy
MH  - High-Throughput Screening Assays/*methods/standards
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods/standards
MH  - *RNA, Small Interfering
OTO - NOTNLM
OT  - FISH
OT  - bDNA
OT  - mRNA
OT  - qRT-PCR
OT  - siRNA
EDAT- 2020/10/06 06:00
MHDA- 2022/02/19 06:00
CRDT- 2020/10/05 08:40
PHST- 2020/10/06 06:00 [pubmed]
PHST- 2022/02/19 06:00 [medline]
PHST- 2020/10/05 08:40 [entrez]
AID - S2472-5552(22)06671-0 [pii]
AID - 10.1177/2472555220960045 [doi]
PST - ppublish
SO  - SLAS Discov. 2021 Feb;26(2):281-291. doi: 10.1177/2472555220960045. Epub 2020 Oct 
      5.