Journal: STAR Protocols

Article Title: Label and quantify mRNA molecules in live cell experiments using SunRISER and dNEMO

doi: 10.1016/j.xpro.2022.101630

Figure Lengend Snippet: Schematic of components and plasmid combinations for typical SunRISER mRNA-labeling experiments (A) mRNA and protein components for SunRISER labeling experiments. (B) Schematics of SunRISER variants SRv.1 (top), SRv.1.1 (center), and SRv.1.2 (bottom) for fluorescence signal amplification. The mRNA transcript (black) is tagged at 3′ UTR with PP7 stem loops (blue). In the first stage of signal amplification, each stem loop can be bound by two PCP coat proteins (yellow) fused to a SunTag GCN4 peptide array (orange). In the second stage of signal amplification, GFP (green) is recruited through antibody-peptide-specific binding between scFv (gray) and GCN4 epitopes. (C) Plasmid maps of the SunRISER two-plasmid (2P) variants consisting of detection plasmids (left) and protein plasmids for SRv.1-2P (top), SRv.1.1-2P (center), and SRv.1.2-2P (bottom). Using the indicated plasmids from Addgene, the GOI CDS for SRv.1 (top, left) is CFP and the GOI CDS for SRv.1.1 and SRv.1.2 (center and bottom, left) is mCherry. Although the 2P variants of SunRISER are simpler to work with, SunRISER variants using three plasmids as described previously in can be used as outlined previously and in Table 1 .

Article Snippet: Variation 2 – SRv.1-2P , Phage-cmv-CFP-24×pp7 (Addgene #40652) , cmv-sfgfp-gb1-scAB-ubc-nls-pcp-5×SunTag (Addgene #185800) , .

Techniques: Plasmid Preparation, Labeling, Fluorescence, Amplification, Peptide Microarray, Binding Assay

Journal: STAR Protocols

Article Title: Label and quantify mRNA molecules in live cell experiments using SunRISER and dNEMO

doi: 10.1016/j.xpro.2022.101630

Figure Lengend Snippet: SunRISER plasmid variants

Article Snippet: Variation 2 – SRv.1-2P , Phage-cmv-CFP-24×pp7 (Addgene #40652) , cmv-sfgfp-gb1-scAB-ubc-nls-pcp-5×SunTag (Addgene #185800) , .

Techniques: Plasmid Preparation

Journal: STAR Protocols

Article Title: Label and quantify mRNA molecules in live cell experiments using SunRISER and dNEMO

doi: 10.1016/j.xpro.2022.101630

Figure Lengend Snippet: SunRISER-labeled mRNAs appear as diffraction-limited spots (A) Image of theoretical point spread functions (PSFs) for diffraction limited signals. The simulated image was generated as described previously (see ( Kowalczyk et al., 2021 )). (B and C) Representative maximum intensity projections of HeLa cells transfected with SunRISER SRv.1-2P and detection plasmid CFP-24×PP7 (top images). Bottom images represent detail of fluorescence images as indicated, with blue circles representing spots detected by dNEMO analysis. In good labeling conditions, (B) mRNA molecules labeled with SunRISER appear as diffraction-limited spots comparable to those generated in the simulated image. In poor labeling (C) fluorescent structures are larger than diffraction-limited objects and do not represent single mRNA molecules. Large objects also show evidence of oversegmentation where a single fluorescent structure is detected as multiple spots. Scale bars: 10 μm.

Article Snippet: Variation 2 – SRv.1-2P , Phage-cmv-CFP-24×pp7 (Addgene #40652) , cmv-sfgfp-gb1-scAB-ubc-nls-pcp-5×SunTag (Addgene #185800) , .

Techniques: Labeling, Generated, Transfection, Plasmid Preparation, Fluorescence

Journal: STAR Protocols

Article Title: Label and quantify mRNA molecules in live cell experiments using SunRISER and dNEMO

doi: 10.1016/j.xpro.2022.101630

Figure Lengend Snippet: Quantification of SunRISER-labeled mRNAs in time-lapse live-cell images with dNEMO (A) Maximum intensity projection of HeLa cells transfected with SunRISER SRv.1-2P and detection plasmid CFP-24×PP7. Cells were imaged every 10 min for 24 h. Scale bar: 10 μm. (B) SunRISER-labeled mRNAs detected by dNEMO (blue or orange circles) and associated with cells segmented using Cellpose. (C) Time-courses for the number of mRNA molecules identified within the 2 cells shown in (A). (D) Example results file generated by dNEMO and output to excel. Features are collected for every spot, and spots within each single cell are separated by tabs.

Article Snippet: Variation 2 – SRv.1-2P , Phage-cmv-CFP-24×pp7 (Addgene #40652) , cmv-sfgfp-gb1-scAB-ubc-nls-pcp-5×SunTag (Addgene #185800) , .

Techniques: Labeling, Transfection, Plasmid Preparation, Generated

Journal: STAR Protocols

Article Title: Label and quantify mRNA molecules in live cell experiments using SunRISER and dNEMO

doi: 10.1016/j.xpro.2022.101630

Figure Lengend Snippet: Spot detection settings and operations in dNEMO (A) The dNEMO software interface with an open image of HeLa cells transfected with SunRISER SRv.1-2P and detection plasmid CFP-24×PP7. Highlighted within the ‘Spot Filter’ panel (upper-right) are critical user operations: the ‘Wavelet Threshold’ value; the detection of spots over the currently displayed image (‘Test Detect’ buttons); and the generation of a keyframe of the current detection settings (‘Create Keyframe’ button). (B) Settings GUI accessible in dNEMO which holds user-defined parameters for spot detection. Highlighted are the ‘Frame Limit’ parameter (upper left) and the ‘Wavelet Level’ parameter (upper right). (C) Screenshot of the ‘Keyframes’ panel after the spot detection operation has completed operating over the time-lapse images.

Article Snippet: Variation 2 – SRv.1-2P , Phage-cmv-CFP-24×pp7 (Addgene #40652) , cmv-sfgfp-gb1-scAB-ubc-nls-pcp-5×SunTag (Addgene #185800) , .

Techniques: Software, Transfection, Plasmid Preparation