Detecting mRNA Expression via FISH

All fluorescence in situ hybridisation (FISH) experiments are performed against a particular mRNA of interest, thereby allow detection of the mRNA expression. FISH was performed as described in King and Newmark, 2013 (link) and previously reported sequences were used for riboprobe synthesis of smedwi-1 (Abnave et al., 2017 (link)). The antibodies used for immunostaining were anti-H3P (phosphorylated serine 10 on histone H3; Millipore; 09–797; 1:1000 dilution Abnave et al., 2017 (link)), anti-poly (ADP) ribose (Shibata et al., 2016 (link)) (PAR) monoclonal antibody (1:250) (Santacruz, clone 10H), and anti-TUD1 (1:250 dilution, based on Solana et al., 2009 (link)). Anti-rabbit-HRP (H3P and TUD-1) and anti-mouse-HRP (PAR) (1:2000 dilution) secondary antibodies were used followed by tyramide signal amplification for FISH and immunostaining as described in King and Newmark, 2013 (link).

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Sahu S., Sridhar D., Abnave P., Kosaka N., Dattani A., Thompson J.M., Hill M.A, & Aboobaker A. (2021). Ongoing repair of migration-coupled DNA damage allows planarian adult stem cells to reach wound sites. eLife, 10, e63779.

Publication 2021

anti-H3P

Antibodies Clone Dilution Fluorescence in situ hybridisation Histone h3 Monoclonal antibody Mouse Mrna Poly adp ribose Rabbit Serine Synthesis

Corresponding Organization :

Other organizations : University of Oxford, CRUK/MRC Oxford Institute for Radiation Oncology

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Variable analysis

independent variables

MRNA of interest

dependent variables

MRNA expression

control variables

All FISH experiments were performed against a particular mRNA of interest
Antibody dilutions used for immunostaining were specified

controls

Previously reported sequences were used for riboprobe synthesis of smedwi-1

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