4.7 Article

Multimodal detection of protein isoforms and nucleic acids from mouse pre-implantation embryos

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NATURE PROTOCOLS
卷 16, 期 2, 页码 -

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NATURE RESEARCH
DOI: 10.1038/s41596-020-00449-2

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资金

  1. National Institutes of Health Training Grant [5T32GM008155-29]
  2. California Institute for Regenerative Medicine Predoctoral Fellowship
  3. Obra Social 'la Caixa' Fellowship
  4. University of California, Berkeley Siebel Scholarship
  5. National Defense Science and Engineering Graduate Fellowship
  6. National Science Foundation CAREER Award [CBET-1056035]
  7. National Institutes of Health [R01CA203018, R01GM114414, R01CA139067, R21HD088885, K99-HHD096108-01, CA192636-03]
  8. Howard Hughes Medical Institute [55108532 HHMI]
  9. Bakar Fellow Award at UC Berkeley
  10. American Cancer Society

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A novel technique (snapBlot) has been introduced for simultaneous measurement of protein isoforms and nucleic acids from the same embryo, with the potential to uncover the regulatory mechanisms of nucleic acids in mammalian embryo development.
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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