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Mouse preimplantation embryos lack zygotically produced activin A. The results of ddPCR analysis of Inhba and <t>Inhbb</t> mRNA levels in wild-type MII oocytes, zygotes, 2-cell, 4-cell, 8-cell, morula-, and blastocyst-stage embryos (pooled samples of 26 to 70 embryos per stage; see Methods section) and a positive control—wild-type ESCs. The graphs illustrate the fluorescent amplitude observed for individual transcripts: the reference housekeeping gene Actb (β-actin gene) and Inhba (activin A gene) or Inhbb (activin B gene). Dots in the upper cluster represent positive droplets in which amplification of the target fragment occurred, while dots in the lower cluster correspond to the negative droplets without a PCR product of: (A) Inhba in ESCs, oocytes, zygotes, and embryos developed in vivo; (B) Inhba in ESCs and embryos developed in vitro; (C) Inhbb in ESCs, oocytes, zygotes, and embryos developed in vivo; (D) Inhbb in ESCs and embryos developed in vitro.
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Mouse preimplantation embryos lack zygotically produced activin A. The results of ddPCR analysis of Inhba and <t>Inhbb</t> mRNA levels in wild-type MII oocytes, zygotes, 2-cell, 4-cell, 8-cell, morula-, and blastocyst-stage embryos (pooled samples of 26 to 70 embryos per stage; see Methods section) and a positive control—wild-type ESCs. The graphs illustrate the fluorescent amplitude observed for individual transcripts: the reference housekeeping gene Actb (β-actin gene) and Inhba (activin A gene) or Inhbb (activin B gene). Dots in the upper cluster represent positive droplets in which amplification of the target fragment occurred, while dots in the lower cluster correspond to the negative droplets without a PCR product of: (A) Inhba in ESCs, oocytes, zygotes, and embryos developed in vivo; (B) Inhba in ESCs and embryos developed in vitro; (C) Inhbb in ESCs, oocytes, zygotes, and embryos developed in vivo; (D) Inhbb in ESCs and embryos developed in vitro.
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Mouse preimplantation embryos lack zygotically produced activin A. The results of ddPCR analysis of Inhba and <t>Inhbb</t> mRNA levels in wild-type MII oocytes, zygotes, 2-cell, 4-cell, 8-cell, morula-, and blastocyst-stage embryos (pooled samples of 26 to 70 embryos per stage; see Methods section) and a positive control—wild-type ESCs. The graphs illustrate the fluorescent amplitude observed for individual transcripts: the reference housekeeping gene Actb (β-actin gene) and Inhba (activin A gene) or Inhbb (activin B gene). Dots in the upper cluster represent positive droplets in which amplification of the target fragment occurred, while dots in the lower cluster correspond to the negative droplets without a PCR product of: (A) Inhba in ESCs, oocytes, zygotes, and embryos developed in vivo; (B) Inhba in ESCs and embryos developed in vitro; (C) Inhbb in ESCs, oocytes, zygotes, and embryos developed in vivo; (D) Inhbb in ESCs and embryos developed in vitro.
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Mouse preimplantation embryos lack zygotically produced activin A. The results of ddPCR analysis of Inhba and <t>Inhbb</t> mRNA levels in wild-type MII oocytes, zygotes, 2-cell, 4-cell, 8-cell, morula-, and blastocyst-stage embryos (pooled samples of 26 to 70 embryos per stage; see Methods section) and a positive control—wild-type ESCs. The graphs illustrate the fluorescent amplitude observed for individual transcripts: the reference housekeeping gene Actb (β-actin gene) and Inhba (activin A gene) or Inhbb (activin B gene). Dots in the upper cluster represent positive droplets in which amplification of the target fragment occurred, while dots in the lower cluster correspond to the negative droplets without a PCR product of: (A) Inhba in ESCs, oocytes, zygotes, and embryos developed in vivo; (B) Inhba in ESCs and embryos developed in vitro; (C) Inhbb in ESCs, oocytes, zygotes, and embryos developed in vivo; (D) Inhbb in ESCs and embryos developed in vitro.
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Mouse preimplantation embryos lack zygotically produced activin A. The results of ddPCR analysis of Inhba and <t>Inhbb</t> mRNA levels in wild-type MII oocytes, zygotes, 2-cell, 4-cell, 8-cell, morula-, and blastocyst-stage embryos (pooled samples of 26 to 70 embryos per stage; see Methods section) and a positive control—wild-type ESCs. The graphs illustrate the fluorescent amplitude observed for individual transcripts: the reference housekeeping gene Actb (β-actin gene) and Inhba (activin A gene) or Inhbb (activin B gene). Dots in the upper cluster represent positive droplets in which amplification of the target fragment occurred, while dots in the lower cluster correspond to the negative droplets without a PCR product of: (A) Inhba in ESCs, oocytes, zygotes, and embryos developed in vivo; (B) Inhba in ESCs and embryos developed in vitro; (C) Inhbb in ESCs, oocytes, zygotes, and embryos developed in vivo; (D) Inhbb in ESCs and embryos developed in vitro.
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Mouse preimplantation embryos lack zygotically produced activin A. The results of ddPCR analysis of Inhba and Inhbb mRNA levels in wild-type MII oocytes, zygotes, 2-cell, 4-cell, 8-cell, morula-, and blastocyst-stage embryos (pooled samples of 26 to 70 embryos per stage; see Methods section) and a positive control—wild-type ESCs. The graphs illustrate the fluorescent amplitude observed for individual transcripts: the reference housekeeping gene Actb (β-actin gene) and Inhba (activin A gene) or Inhbb (activin B gene). Dots in the upper cluster represent positive droplets in which amplification of the target fragment occurred, while dots in the lower cluster correspond to the negative droplets without a PCR product of: (A) Inhba in ESCs, oocytes, zygotes, and embryos developed in vivo; (B) Inhba in ESCs and embryos developed in vitro; (C) Inhbb in ESCs, oocytes, zygotes, and embryos developed in vivo; (D) Inhbb in ESCs and embryos developed in vitro.

Journal: Biology of Reproduction

Article Title: Maternal/zygotic knockout and droplet digital polymerase chain reaction analysis question the role of activin A in preimplantation mouse embryo development †

doi: 10.1093/biolre/ioaf189

Figure Lengend Snippet: Mouse preimplantation embryos lack zygotically produced activin A. The results of ddPCR analysis of Inhba and Inhbb mRNA levels in wild-type MII oocytes, zygotes, 2-cell, 4-cell, 8-cell, morula-, and blastocyst-stage embryos (pooled samples of 26 to 70 embryos per stage; see Methods section) and a positive control—wild-type ESCs. The graphs illustrate the fluorescent amplitude observed for individual transcripts: the reference housekeeping gene Actb (β-actin gene) and Inhba (activin A gene) or Inhbb (activin B gene). Dots in the upper cluster represent positive droplets in which amplification of the target fragment occurred, while dots in the lower cluster correspond to the negative droplets without a PCR product of: (A) Inhba in ESCs, oocytes, zygotes, and embryos developed in vivo; (B) Inhba in ESCs and embryos developed in vitro; (C) Inhbb in ESCs, oocytes, zygotes, and embryos developed in vivo; (D) Inhbb in ESCs and embryos developed in vitro.

Article Snippet: Droplet digital polymerase chain reaction was performed in 20 μl reactions containing 10 μl ddPCR Supermix for Probes (Bio-Rad), 1 μl of specific TaqMan probes for β-actin (Mm01205647_g1), activin A (Mm00434339_m1) or activin B (Mm03023992_m1) (Applied Biosystem), 3 μl of PCR-grade water and 6 μl of cDNA.

Techniques: Produced, Positive Control, Amplification, In Vivo, In Vitro