After generating cDNA earlier today from Noah’s Crassostrea gigas (Pacific oyster) RNA (isolated 20230712), I ran qPCRs using the pooled cDNA sample to test out some primers that Steven pulled from the freezer.
| SRID | PrimerName | Organism |
|---|---|---|
| 1614 | Defensin_FWD | C.gigas |
| 1613 | Defensin_REV | C.gigas |
| 1626 | HSP70c_FWD | O.lurida |
| 1625 | HSP70c_REV | O.lurida |
| 256 | IL-17 internal RV | C.gigas |
| 255 | IL-17 internal FW | C.gigas |
| 1630 | TLR2.1_FWD | O.lurida |
| 1629 | TLR2.1_REV | O.lurida |
NOTE: We did not have the above info prior to running. Only discovered after qPCR completed that two primer sets were for a different species!
All reactions were run with 2x SsoFast EvaGreen Master Mix (BioRad), using 1uL of cDNA. See qPCR Report in the RESULTS section below for cycling params, etc.
qPCR calcs:
- 20230713 - qPCR Calcs Noah gigas ctenidia pooled cDNA (Google Sheet)
RESULTS
qPCR Data file (QPCRD - CFX Maestro required)
qPCR Report (PDF)
Unsurprisingly, the two primer sets designed for Ostrea lurida (Olympia oyster) do not amplify.
Defensin comes up very late (~39 Cq), while IL-17 comes up earlier (~32Cq). The melt curves look good, although there might be a slight hump present in the IL-17 reactions… See plots below.
Amplification Plots
- Defensin in GREEN
- IL-17 in BLACK
Melt Curves
- Defensin in GREEN
- IL-17 in BLACK
