MilliporeSigma

Single molecule imaging of RNA in situ.

Methods in molecular biology (Clifton, N.J.) (2011-03-25)
Mona Batish, Arjun Raj, Sanjay Tyagi
ABSTRACT

This protocol describes a method to image individual mRNA molecules in situ. About 50 oligonucleotides complementary to different regions of a target mRNA species are used simultaneously. Each probe is labeled with a single fluorescent moiety. When these probes bind to their target, each mRNA molecule becomes so intensely fluorescent that it can be seen as a fine fluorescent spot. Several different mRNA species can be detected in multiplex imaging using differently colored probe sets for each species. An automated image-processing program is used to count the number of mRNA molecules of each species that are expressed in each cell, thus yielding single-cell gene expression profiles.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dextran sulfate sodium salt from Leuconostoc spp., for molecular biology, average Mw >500,000 (dextran starting material), contains 0.5-2% phosphate buffer
Sigma-Aldrich
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, 1-2 mg/mL in Tris-buffered saline, 0.2 μm filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
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