Abstract
Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not yet clear. Challenges arise in measuring protein isoforms and nucleic acids from the same single embryos and blastomeres. Here we present a multimodal technique for performing same-embryo nucleic acid and protein isoform profiling (single-embryo nucleic acid and protein profiling immunoblot, or snapBlot). The method integrates protein isoform measurement by fractionation polyacrylamide gel electrophoresis (fPAGE) with off-chip analysis of nucleic acids from the nuclei. Once embryos are harvested and cultured to the desired stage, they are sampled into the snapBlot device and subjected to fPAGE. After fPAGE, ‘gel pallets’ containing nuclei are excised from the snapBlot device for off-chip nucleic acid analyses. fPAGE and nuclei analyses are indexed to each starting sample, yielding same-embryo multimodal measurements. The entire protocol, including processing of samples and data analysis, takes 2–3 d. snapBlot is designed to help reveal the mechanisms by which embryo-specific nucleic acid modifications to both genomic DNA and messenger RNA orchestrate the growth and development of mammalian embryos.
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Data availability
Source data are provided with this paper.
Code availability
MATLAB analysis scripts are available in Supplementary Data 1. These analysis scripts (particularly intProf.m, fitPeaks.m and goodProfiles.m) have been adapted from those posted as part of the summit code for single-cell western blot analysis, which can be found at https://github.com/herrlabucb/summit.
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Acknowledgements
We acknowledge members and alumni of the Herr Lab for useful discussions. Partial infrastructure support was provided by the QB3 Biomolecular Nanofabrication Center. This research was performed under a National Institutes of Health Training Grant awarded to the UCB/USCF Graduate Program in Bioengineering (5T32GM008155-29 to E.R.-C. and A.G.), a California Institute for Regenerative Medicine Predoctoral Fellowship (to E.R.-C.), an Obra Social ‘la Caixa’ Fellowship (to E.R.-C.), a University of California, Berkeley Siebel Scholarship (to E.R.-C.), a National Defense Science and Engineering Graduate Fellowship (to A.G.), a National Science Foundation CAREER Award (CBET-1056035 to A.E.H.), National Institutes of Health grants R01CA203018 (to A.E.H.) and R01GM114414, R01CA139067 and R21HD088885 (to L.H.), a Howard Hughes Medical Institute (55108532 HHMI) Faculty Scholar Award (to L.H.), a Bakar Fellow Award at UC Berkeley (to L.H.), a Research Scholar Award from the American Cancer Society (to L.H.), an F32 Postdoctoral Fellowship from the National Institutes of Health (CA192636-03 to A.J.M.) and a K99 Career Transition Award from the National Institutes of Health (K99-HHD096108-01 to A.J.M.).
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Contributions
E.R.-C. conceived the idea for the snapBlot assay. E.R.-C. and A.G. performed immunoblotting experiments and analyzed immunoblotting data. A.J.M. collected, cultured and handled mouse embryos, performed RT–qPCR experiments and analyzed RT–qPCR data. All authors wrote the manuscript.
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The authors are inventors on pending patents related to snapBlot assays.
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Key reference using this protocol:
Rosàs-Canyelles, E. et al. Sci. Adv. 6, eaay1751 (2020): https://doi.org/10.1126/sciadv.aay1751
Supplementary information
Supplementary Information
Supplementary Figs. 1–4.
Supplementary Data 1
MATLAB script for protein image analysis.
Supplementary Data 2
CAD photomask design file for Si/Su-8 wafer fabrication.
Supplementary Video 1
Video of snapBlot device fabrication.
Supplementary Video 2
Video of embryos loaded into snapBlot device microwells using a mouth pipette assembly.
Source data
Source Data Figs. 6–8
Unprocessed fluorescence microarray scan of immunoprobed snapBlot, Figs. 6–8.
Source Data Figs. 6–8
Excel file with data for Figs. 6–8
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Rosàs-Canyelles, E., Modzelewski, A.J., Geldert, A. et al. Multimodal detection of protein isoforms and nucleic acids from mouse pre-implantation embryos. Nat Protoc 16, 1062–1088 (2021). https://doi.org/10.1038/s41596-020-00449-2
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DOI: https://doi.org/10.1038/s41596-020-00449-2
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