qPCR - C.gigas cDNA Primer Tests for Noah’s Heat-Mechanical Stress Project

qPCR
Crassostrea gigas
cDNA
Noah
Pacific oyster
2023
Author

Sam White

Published

July 13, 2023

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:

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

qPCR amplification plots for defensin (green) and Il-17 (black). Defensin comes up late, at ~39Cq. IL-17 comes up earlier at ~32Cq

Melt Curves

  • Defensin in GREEN
  • IL-17 in BLACK

qPCR melt plots for defensin (green) and Il-17 (black).