Abstract
RNA labeling in situ has enormous potential to visualize transcripts and quantify their levels in single cells, but it remains challenging to produce high levels of signal while also enabling multiplexed detection of multiple RNA species simultaneously. Here, we describe clampFISH 2.0, a method that uses an inverted padlock design to efficiently detect many RNA species and exponentially amplify their signals at once, while also reducing the time and cost compared with the prior clampFISH method. We leverage the increased throughput afforded by multiplexed signal amplification and sequential detection to detect 10 different RNA species in more than 1 million cells. We also show that clampFISH 2.0 works in tissue sections. We expect that the advantages offered by clampFISH 2.0 will enable many applications in spatial transcriptomics.
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Data availability
All imaging and RNA sequencing data are publicly available on Dropbox https://www.dropbox.com/sh/q51kmcphoyi9yi3/AAB4g1a6ODDHaphsvbmBJAy-a?dl=0, with a description of how data were associated with each figure in the README.docx and ExperimentList.xlsx files. RNA sequencing data are also deposited on Gene Expression Omnibus (GEO accession GSE211491). Source data are provided with this paper.
Code availability
All MATLAB data analysis code, including repositories for rajlabimagetools, dentist2, pixyDuck, bfmatlab, Cellpose, and scripts for raw data processing, image data extraction and plotting, is publicly available on Dropbox (https://www.dropbox.com/sh/q51kmcphoyi9yi3/AAB4g1a6ODDHaphsvbmBJAy-a?dl=0), with instructions in the README.docx file and also on GitHub (https://github.com/iandarr/clampFISH2allcode). Code was run with MATLAB R2021a (64-bit, maci64), available from www.mathworks.com.
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Acknowledgements
The authors thank A. Coté and I.A. Mellis for helpful input on image analysis; the ENCODE Consortium and the laboratory of J.M. Cherry for RNA-seq datasets used for probe design; the Penn Center for Musculoskeletal Disorders Histology Core (P30 AR069619) for their guidance on tissue cryo-sectioning; and the Wistar Institute’s Histotechnology facility (supported by Cancer Center Support Grant P30 CA010815) for processing the FFPE tissue samples. The authors acknowledge support from the National Institutes of Health (NIH) grants F30 CA236129, T32 GM007170 and T32 HG000046 (to B.L.E.); a Career Award at the Scientific Interface from BWF and the Schmidt Science Fellowship in partnership with the Rhodes Trust (to Y.G.); training grants NIH F30 HG010822, NIH T32 DK007780 and NIH T32 GM007170 (to C.L.J.); NIH K00-CA-212437-02 (to A.K.); the Chan Zuckerberg Initiative (to S.H.R.); R01CA174746 and R01CA207935 (to M.E.F. and A.T.W); a Team Science Award from the Melanoma Research Alliance and P01 CA114046 (to A.T.W.); NIH grants RO1 CA238237, U54 CA224070, PO1 CA114046, P50CA174523 and the Dr Miriam and Sheldon G. Adelson Medical Research Foundation (to M.H.); 5-U2C-CA-233285-04, NIH 4DN U01 HL129998 and NIH 4DN U01DK127405 (to I.D. and A.R.); and the NIH Center for Photogenomics RM1 HG007743, NIH Director’s Transformative Research Award R01 GM137425, and the Penn Epigenetics Institute (to A.R.).
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Authors and Affiliations
Contributions
I.D. designed, performed and analyzed all experiments (supervised by A.R.). B.L.E. and C.L.J. assisted with FISH protocol development. Y.G. derived and isolated the WM989 A6-G3 RC4 cell line, assisted with tissue sectioning, assisted with cell culture and performed all RNA sequencing. B.L.E. derived and isolated the WM989 A6-G3 H2B-GFP cell line. A.K., C.L.J., B.L.E. and S.H.R. assisted with probe synthesis protocol development. J.L. performed cell segmentation for the amplifier screen experiment. G.M.A., M.E.F. and A.T.W. performed the mouse experiment with WM989-A6-G3-Cas9-5a3 cells and provided dissected tumor samples for the fresh frozen tissue experiment. M.X. and M.H. performed the mouse experiment with patient-derived tissue and prepared the samples up to paraffin embedding.
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Competing interests
I.D., B.L.E. and A.R. have filed a patent application related to this work. A.R. receives patent royalties from LGC/Biosearch Technologies related to Stellaris mRNA FISH products. A.R. is on the scientific advisory board of Spatial Genomics. A.T.W. is on the Board of Directors at ReGAIN therapeutics. All other authors have no competing interests.
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Extended data
Extended Data Fig. 1 clampFISH 2.0 amplifies GFP RNA FISH signal with high specificity.
Images of GFP clampFISH 2.0 spots in drug-naive H2B-GFP WM989 A6-G3 cells (top) and vemurafenib-resistant WM989 A6-G3 RC4 cells (bottom) with a 20 nucleotide secondary-targeting readout probe (labeled with Atto 647N) and conventional single-molecule RNA FISH probes (labeled with Alexa 555) targeting different regions of the same RNA. Shown are maximum intensity projections of 9 z-planes at 60X magnification. Shown are data using amplifier set 1, where 15 amplifier sets were tested in total. The experiment was performed once. See Supplementary Fig. 2 for co-localization quantification of all amplifier sets. For details, see Supplementary Methods description of amplifier screen experiment. As expected, we observed bright GFP clampFISH 2.0 spot counts in cells with nuclear-localized GFP signal, but not in cells without the H2B-GFP construct.
Extended Data Fig. 2 clampFISH 2.0 amplifies EGFR RNA FISH signal with high accuracy.
Images of EGFR clampFISH 2.0 spots in drug-naive H2B-GFP WM989 A6-G3 cells (top) and vemurafenib-resistant WM989 A6-G3 RC4 cells (bottom) with a 20 nucleotide secondary-targeting readout probe (labeled with Atto 647N) and conventional single-molecule RNA FISH probes (labeled with Cy3) targeting different regions of the same RNA. Shown are maximum intensity projections of 9 z-planes at 60X magnification. Shown are data using amplifier set 1, where 15 amplifier sets were tested in total. The experiment was performed once. For details, see Supplementary Methods description of amplifier screen experiment. As expected from bulk RNA sequencing data, we observed many more EGFR clampFISH 2.0 spots in the vemurafenib-resistant cells than in the drug-naive cells.
Extended Data Fig. 3 clampFISH 2.0 eliminates the bright, non-specific fluorescent spots that were observed in clampFISH 1.0.
Top left: clampFISH 1.0 targeting GFP in WM983b-GFP melanoma cells, amplified to round 6 with amplifier probes containing an internal Cy5 dye and imaged at 20X with a 3 second exposure time using a cooled CCD camera with a 13μm pixel size (image from Rouhanifard et al. 2018; see that paper for further details). The two arrows point to two of the non-specific spots. Top right: clampFISH 2.0 targeting GFP in a mixed population of cells (a majority of WM989 A6-G3 H2B-GFP cells and fewer WM989 A6-G3 RC4 cells), amplified to round 8 with readout probes labeled with Atto 647N and imaged at 20X with a 1 second exposure time using a sCMOS camera with a 6.5μm pixel size. Image shown is from the present work’s ‘pooled amplifier experiment’, which was performed once. For all experiments performed in this work, we observed similar results to those depicted here. Bottom: zoomed-in views of the top images. We found that we could eliminate the bright non-specific spots by introducing a number of centrifugation steps to both the primary probe and amplifier probe synthesis protocols. To perform this step, we centrifuge the solution in 1.5 mL tubes at 17,000 g for 20 minutes and transfer the top portion of the solution to a new tube and discarded the bottom portion. We perform this step twice after the enzymatic steps are complete, and once after ethanol precipitation (see Supplementary Fig. 1). Additionally, we found that by adding the centrifugation step to completed clampFISH 1.0 probe solutions, we could similarly reduce the non-specific spots seen in that method.
Extended Data Fig. 4 clampFISH 2.0 amplifies signal exponentially.
(a) In an amplification characterization experiment, we performed clampFISH 2.0 with amplification to varying rounds (round 1, 2, 4, 6, 8, and 10) and then hybridized four readout probes to measure the spot intensities, with the median intensity from rounds 2, 4, 6, 8 and 10 fit to an exponential curve (labeled values are median intensities). We found that every round the spot intensities grew by a factor of 1.457, 1.586, 1.406, and 1.527 for each probe set respectively. With a hypothetical 2:1 binding ratio of each amplifier probe to the previous probe, these factors suggest a per-probe binding efficiency of 73%, 79%, 70%, and 76%, respectively. (b) Replicate 2 of the same experiment as in (a), where the spot intensities grew by a factor of 1.525, 1.678, 1.496, and 1.628, suggesting per-probe binding efficiencies of 76%, 84%, 75%, and 81%, respectively. For spot counts associated with each condition in panels a-b, see Supplementary Table 12. Circles are median values and bounds of boxes are 25th and 75th percentiles.
Extended Data Fig. 5 clampFISH 2.0 spot sizes remain similar throughout the rounds of amplification.
(a) Cropped images of spots from UBC clampFISH 2.0 with readout probes in Atto 488 at varying levels of amplification (from left to right: round 1, 2, 4, 6, 8, and 10) imaged with a 100X/1.45NA objective (65 nm pixel sizes). A spot with a representative (median) fitted amplitude was chosen for display. The minimum intensity (black) and maximum intensity (white) used for contrasting are shown below the images. Contrasting is applied equally to all images (top row) or set to each image’s minimum and maximum values (bottom row). (b) ClampFISH 2.0 was performed to varying rounds of amplification using primary probes targeting UBC mRNA, amplifier set 9, and readout probes labeled in Atto 488 (top panels) or using primary probes targeting MITF, amplifier set 12, and readout probes labeled in Atto 647N (bottom panels). Samples were imaged with a 100X/1.45NA objective (65 nm pixel sizes) and each called spot was fit at its maximal-intensity z-plane to a 2D Gaussian distribution. Shown are the standard deviation of each spot’s Gaussian fit (left panels), amplitude of each spot’s Gaussian fit normalized to the round 1 median amplitude (middle panels), and each segmented cell’s spot count (right panels). For the left and middle panels, circles and numbers shown are median values and bounds of boxes are 25th and 75th percentiles. For UBC data, n = 923, 1437, 1968, 1737, 2251, 846 spots and for MITF data n = 1206, 1219, 994, 1634, 1450, and 930 for rounds 1,2,4,6,8, and 10, respectively. For the right panel, circles are median values, bounds of boxes are 25th and 75th percentiles, and whiskers extend to non-outlier minima and maxima, where data falling more than 1.5 times the interquartile range beyond the box bounds are considered outliers. Theoretical standard deviations of Gaussian approximations of diffraction-limited spots (0.21λ/NA; with paraxial optics assumptions) with wavelengths at the midpoints of the emission filters (535 nm for Cy3; 667 nm for Atto 647N) are 77.5 nm (Cy3) and 96.6 nm (Atto 647N).
Extended Data Fig. 6 clampFISH 2.0 spot sizes are similar to conventional single-molecule RNA FISH spot sizes.
Conventional single-molecule RNA FISH (smFISH) spot sizes are compared to clampFISH 2.0 spots imaged on the same day (experiment 2; see method section for description of positive control for ‘one-pot’ experiment) and to clampFISH 2.0 spots in a previous experiment (experiment 1, also depicted in Extended Data Fig. 5; see method section description of ‘amplification characterization’ experiment). We imaged the samples with a 100X/1.45NA objective at 1×1 camera binning (65 nm pixel size) and fit the pixel values in the neighborhood of each spot to a 2D Gaussian distribution. Left: standard deviation of Gaussian-fitted spots for UBC smFISH labeled in Atto 488 and UBC clampFISH 2.0 amplified to round 1 or round 4 with readout probes labeled in Atto 488. Right: standard deviation of Gaussian-fitted spots for TOP2A smFISH labeled in Atto 647N and MITF clampFISH 2.0 amplified to round 1 or round 4 with readout probes labeled in Atto 647N. Values shown are the median standard deviations. For the left and right panels, circles and numbers shown are median values and bounds of boxes are 25th and 75th percentiles. For Atto 488 data (left panel), n = 1053, 923, and 1968 (from left to right) and for Atto 647N data (right panel) n = 1875, 1230, 2254, 1206, and 994 (from left to right).
Extended Data Fig. 7 clampFISH 2.0 quantifies RNA spot counts at 10X magnification.
Depicting the same data as in Fig. 2b, but with clampFISH 2.0 spots imaged at 10X magnification. We performed clampFISH 2.0 for 10 genes, amplified the 10 scaffolds in parallel to round 8, then added a single pair of readout probes to label a scaffold corresponding to AXL (left; in drug-resistant WM989 A6-G3 RC4 cells), EGFR (middle; in drug-resistant WM989 A6-G3 RC4 cells), or DDX58 (right; in drug-naïve WM989 A6-G3 cells). In two biological replicates (top: replicate 1; bottom: replicate 2), we counted spots for clampFISH 2.0 at 10X magnification (y-axis) and conventional single-molecule RNA FISH at 60X magnification (x-axis), which targeted non-overlapping regions of the same RNAs. In replicate 2, imaging at 10X of DDX58 spots before conventional single-molecule RNA FISH was not performed.
Extended Data Fig. 8 Signal from the previous readout cycle is removed after a high-formamide strip.
(a) Example images of clampFISH 2.0 spots at 20X magnification before the readout probe hybridization (top row), after adding readout probes (middle row), and after stripping off readout probes (bottom row). The first three columns are from readout cycle 1, the next three are from readout cycle 2, and the last 4 columns are from readout cycle 3. Each column’s images are from the same channel (with the corresponding readout probe dye indicated), exposure time (as indicated in milliseconds), and are contrasted identically. (b) Example images as in (a) at a different position on the plate. The experiment was performed twice with similar results.
Extended Data Fig. 9 clampFISH 2.0 scaffolds remain stably bound after multiple rounds of readout stripping and storage at 4 °C for 4 months.
Images of clampFISH 2.0 spots from a 20X objective over readout cycles where we repeatedly use 4 sets of readout probes which label (from top to bottom) AXL, WNT5A, DDX58, and UBC clampFISH 2.0 scaffolds. Column 1: readout cycle 1. Column 2: readout cycle 1, re-imaged after removing the sample from the microscope stage and stored overnight at 4 °C. Column 3: after stripping off readout probes from readout cycle 1. Column 4: readout cycle 4, where we repeat readout cycle 1 after readout cycles 2 and 3 (where different sets of genes were labeled). Column 5: readout cycle 5, performed after storing the sample at 4 °C in 2X SSC for 4 months. DAPI overlay is contrasted separately for each column. Each row of readout cycle 5 (column 5) is contrasted with 180% the intensity range of the first four columns. The cycle 5 signal presumably appeared brighter due to changes in the microscope’s optics during that time frame (for example, greater sample illumination or increased transmission to the sensor). The experiment was performed twice with similar results, except for column 5 data (after 4 months storage) which was performed once. See Supplementary Figs. 14 and 15 for quantification of each experimental replicate.
Extended Data Fig. 10 clampFISH 2.0 detects RNAs in presumptive human cells in tissue.
clampFISH 2.0 was performed in a 6μm fresh frozen tissue section of a dissected tumor, derived from human WM989-A6-G3-Cas9-5a3 cells injected into a mouse and fed chow containing the BRAFV600E inhibitor PLX4720. Shown are stitched maximum intensity projections of 20X image stacks with 5 z-planes at 1.2μm z-step increments. (a) Pink outlines around regions containing mostly presumptive human cells, demarcated based on nuclear morphology, showing DAPI staining alone (left) and DAPI with UBC clampFISH 2.0 signal overlaid (right), where images are from readout cycle 2. (b) clampFISH 2.0 scaffolds for 10 genes were probed across readout cycles 1 (left), 2 (middle), and 3 (right), where the UBC scaffold was probed each round as a positive control. The dyes on each readout probe set were (top to bottom): Atto488, Cy3, Alexa Fluor 594, and Atto647N. The experiment was performed twice with similar results.
Supplementary information
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Supplementary Methods, Supplementary Figs. 1–18
Supplementary Data 1
Source data for Supplementary Figs.
Supplementary Table 1
Supplementary Tables 1–13
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Source Data Fig. 1
Source data for Fig. 1e,f plots
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Source data for Fig. 2b plots
Source Data Fig. 3
Source data for Fig. 3c heatmap
Source Data Extended Data Fig. 4
Source data for amplification characterization plots
Source Data Extended Data Fig. 5
Source data for clampFISH 2.0 spot size plots
Source Data Extended Data Fig. 6
Source data for clampFISH 2.0 versus smFISH spot size plots
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Dardani, I., Emert, B.L., Goyal, Y. et al. ClampFISH 2.0 enables rapid, scalable amplified RNA detection in situ. Nat Methods 19, 1403–1410 (2022). https://doi.org/10.1038/s41592-022-01653-6
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DOI: https://doi.org/10.1038/s41592-022-01653-6
