Fig. 2: AmpFISH facilitates labeling of transcripts with high specificity and sensitivity (a–c) and the effective amplification of fluorescent signals (d–e) in cultured cells.
From: Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization
a A set of primary amplification probes for targeting the EYFP mRNA was used in HeLa cells transiently expressing EYFP. FISH signal (red, signal labeled by Cy5, the third panel from left in (a) and fluorescent protein signal (green signal in EYFP panel, the second panel in (a) were imaged in the same field of view (scale bar: 50 μm). b The four endogenous mRNAs (Actinβ, Lox, Txn1, and Sla2) were assayed using AmpFISH imaging in NIH3T3 cell lines (scale bar: 20 μm). c The quantitative analysis signal puncta in (b) of the four endogenous mRNAs in NIH3T3 cells of AmpFISH puncta per cell are shown. The mean ± SEM of AmpFISH puncta per cell are shown using red round dots, n(Actinβ) = 20; n(Lox) = 15; n(Txn1) = 15, and n(Sla2) = 19. The four transcripts from the RNAseq dataset (blue line and square dots) were compared. Each dot represents the mean ± SEM (n = 3); FPKM denotes Fragments Per Kilobase Million. These results illustrate the similar relative transcript abundance between the different methods. d Fluorescent signal labeled by TAMRA dye was amplified by AmpFISH in stable NIH3T3/cas9 cells. The confocal imaging for smFISH (unextended probe), primary, secondary, and tertiary amplifications are arranged from left to right, respectively (scale bar: 50 μm). e Comparative analysis of the signal-to-noise ratio of different amplification hierarchies. The intensity of the background signal, from an area adjacent to the area from which the signal was recorded, was subtracted from the intensity of the signal. The signal-to-noise ratio was determined using the formula: (Signal-background)/background.
