Geoduck hemolymph extraction

Developing assay for VTG detection

  • Met with Jose (he has experience with developing Vitellogenin ELISA assays in fish)
    - Options we discussed:
    - ELISA: quantitative
    - Western Blot: not quantitative
    - qPCR: quantitative
    - Calcium assay; calcium correlated with vitellogenin (vitellogenin binds Ca2+)
    - assay for Upstream vitellogenesis inducer? (i.e. estradiol, although Brent says Geoducks likely don’t have this)

    • To develop ELISA:
      • need
        1) isolated vitellogenin to generate standard curve and generate antibody
        • can isolate vitellogenin by:
          • fractionation? It’s large
          • inducing expression
          • cloning and doing bacterial expression
            2) antibody
        • can generate antibody by sending protein (or protein fragment) to commercial antibody lab (they inject rabbits)
    • To develop Western:
      • only need antibody

    • Calcium assay

    • To develop qPCR:
      • need primers
      • need sequence
        • VTG superfamily domain?
          -Align protein sequence to Pgenr genome (tblastn)
          - tblastn web interface finds
          -revtrans on serial cloer to get DNA sequence?
          -tried this and BLASTn web interface finds nothing significant
        • BLAST to Geoduck genome
          • build BLAST index out of Geoduck genome
          • align VTG superfamily domain to genome
          • align protein to genome
      • samples
        • no template
        • non-DNase treated sample or gDNA prep?
        • RNApreps
          • hemocytes from different stages
            • immature
            • early female
            • middle female
            • late female
            • post spawn female
            • male
          • gonad tissue from different stages
            • immature
            • early
            • middle
            • late
            • post spawn
          • hepatopancreas
            • immature
            • early
            • middle
            • late
            • post spawn

VTG Papers