Document Detail


In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay.
MedLine Citation:
PMID:  23348363     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.
Authors:
Irene Weibrecht; Elin Lundin; Sara Kiflemariam; Marco Mignardi; Ida Grundberg; Chatarina Larsson; Björn Koos; Mats Nilsson; Ola Söderberg
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Publication Detail:
Type:  Journal Article     Date:  2013-01-24
Journal Detail:
Title:  Nature protocols     Volume:  8     ISSN:  1750-2799     ISO Abbreviation:  Nat Protoc     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-25     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101284307     Medline TA:  Nat Protoc     Country:  England    
Other Details:
Languages:  eng     Pagination:  355-72     Citation Subset:  IM    
Affiliation:
1] Science for Life Laboratory, Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. [2] [3].
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