Imaging individual mRNA molecules using multiple singly labeled probes

Raj, Arjun; Bogaard, Patrick van den; Rifkin, Scott A.; Oudenaarden, Alexander van; Tyagi, Sanjay

doi:10.1038/nmeth.1253

Cited by 1,855 publications

(2,060 citation statements)

References 15 publications

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“…The proposed RNA cell sorting technique uses flow cytometry to measure the fluorescence of individual cells labeled with a single molecule RNA FISH (smFISH) probe library 13,14 .…”

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confidence: 99%

Transcriptional profiling of cells sorted by RNA abundance

et al. 2014

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We have developed a quantitative technique for sorting cells based on endogenous RNA abundance with a molecular resolution of 10-20 transcripts. We demonstrate efficient and unbiased RNA extraction from transcriptionally sorted cells and report a high fidelity transcriptome measurement of (mouse) induced pluripotent stem cells (iPSCs) isolated from a heterogeneous reprogramming culture. This method is broadly applicable to profiling transcriptionally distinct cellular states without requiring antibodies or transgenic fluorescent proteins.

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“…The proposed RNA cell sorting technique uses flow cytometry to measure the fluorescence of individual cells labeled with a single molecule RNA FISH (smFISH) probe library 13,14 .…”

mentioning

confidence: 99%

Transcriptional profiling of cells sorted by RNA abundance

et al. 2014

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“…In order to define the amplitude of pathway activity we used single molecule fluorescent in situ hybridization (smFISH) to quantitate RNA transcript density of the Hh target genes Ptch1 and Gli1 in individual cells (Figure 1a) (Junker et al, 2014;Raj et al, 2008). Given follicular Hh pathway activity would confound the measurements attributable to loss of Ptch, we restricted our measurements to the interfollicular epidermis (IFE) region of each mouse model.…”

Section: Resultsmentioning

confidence: 99%

“…Probes were coupled to Alexa594 and Cy5 fluorophores as previously described (Lyubimova et al, 2013). Tissue sections were hybridized as previously described (Lyubimova et al, 2013;Raj et al, 2008). Briefly, sections were hybridized with ~0.3 ng/µl of labelled probe sets for each mRNA overnight at 30°C in the dark.…”

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

“…Diffraction-limited dots corresponding to single mRNA molecules were detected in 3D by custom Matlab software using previously described algorithms (Raj et al, 2008). Briefly, the images were first filtered using a three-dimensional Laplacian of Gaussian filter with a width of 15 pixels and a standard deviation of 1.5 pixels.…”

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

“…We then chose the intensity threshold at which the number of connected components was least sensitive to the threshold (Itzkovitz et al, 2012). Individual images (tiles) were taken and stitched together in Matlab software (Raj et al, 2008) …”

Section: A C C E P T E D Accepted Manuscriptmentioning

confidence: 99%

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Patched Receptors Sense, Interpret, and Establish an Epidermal Hedgehog Signaling Gradient

Adolphe

Junker

Lyubimova

et al. 2017

Journal of Investigative Dermatology

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By employing the sensitivity of single molecule fluorescent in situ hybridisation (smFISH) we have precisely quantified the levels and defined the temporal and spatial distribution of Hedgehog signalling activity during embryonic skin development, and uncovered that there is a Hedgehog signalling gradient along the proximal-distal axis of developing hair follicles. In order to explore the contribution of Hedgehog receptors Ptch1 and Ptch2 in establishing the epidermal signalling gradient, we quantitated the level of pathway activity generated in Ptch1 and Ptch1;Ptch2-deficient skin and defined the contribution of each receptor to regulation of the levels of Hedgehog signalling identified in wild-type skin. Moreover, we show that both the cellular phenotype and level of pathway activity featured in Ptch1;Ptch2-deficient cells faithfully recapitulates the Peak level of endogenous Hedgehog signalling detected at the base of developing follicles, where the concentration of endogenous Shh is predicted to be highest.Taken together, these data demonstrate that both Ptch1 and Ptch2 play a crucial role in sensing the concentration of Hedgehog ligand and regulating the appropriate dose-dependent response.

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