Protocols

Flow cytometric analysis of RNA in cells in suspension | Detection of cytokine mRNAs by flow-cytometry | Analysis of T cell activation by using RNA targets | Multiplex flow-cytometric analysis of RNA and proteins

Arrigucci R, Bushkin Y, Radford F, Lakehal K, Vir P, Pine R, Martin D, Sugarman J, Zhao Y, Yap GS, Lardizabal AA, Tyagi S, Gennaro ML (2017) FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry. Nat Protoc 12: 1245-1260.

single-molecule FISH with neurons and tissue culture cells on coverslips

Batish M, Raj A, Tyagi S (2011) Single molecule imaging of RNA in situ. Methods in Molecular Biology 714: 3-13.

Labeling and purification of single-molecule FISH probes | Hybridization with tissue culture cells, yeast, C. elegans embryos, Drosophila melanogaster wing imaginal discs | Fade resistant mounting medium | Image acquisition and analysis

Important tip: Prepare the 0.1M sodium bicarbonate solution (pH 8.3) fresh right before you couple succinimidyl ester derivatives of dyes to probes with amino groups. The pH will be correct without addition of any base. The pH will drift upon storage.

Raj, A and Tyagi, S (2010) Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes, Methods in Enzymology, 472, 365-386.

Tyagi, S (2001) Designing Molecular Beacons

Synthesis, purification and characterization of molecular beacons | Measurements of signal-to-background ratios

Important tip: Use fresh sodium bicarbonate solutions (see above).

Marras SAE, Kramer FR, and Tyagi S (2003) Genotyping single nucleotide polymorphisms with molecular beacons. In "Single Nucleotide Polymorphisms: Methods and Protocols," Kwok PY, ed., Humana Press, Totowa, New Jersey

Manganelli, R, Tyagi, S, and Smith, I (2001) Real Time PCR Using Molecular Beacons, In “Mycobacterium Tuberculosis Protocols”, Parish and Stoker ed., Humana Press Inc., Totowa, New Jersey

Mhlanga MM and Tyagi S. (2006) Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells. Nature Protocols 1, 1392-1396

Construction and cloning of tandemly repeated arrays | Live cell imaging RNAs that harbor tandem repeats | Tracking RNA molecules within nucleus

Bogaard, P.T. C. van den and Tyagi, S (2008) Using Molecular Beacons to Study Dispersalof mRNPs from the Gene Locus. In “The Nucleus, Volume 2: Chromatin, Transcription, Envelope, Proteins, Dynamics and Imaging” Hancock, R, ed., Humana Press, Totowa, New Jersey.