---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: qPCR - C.gigas cDNA Primer Tests for Noah's Heat-Mechanical Stress Project
date: '2023-07-13 12:55'
tags: 
  - qPCR
  - Crassostrea gigas
  - cDNA
  - Noah
  - Pacific oyster
categories: 
  - 2023
  - Miscellaneous
---
After [generating cDNA earlier today](https://robertslab.github.io/sams-notebook/posts/2023/2023-07-13-Reverse-Transcription-C.gigas-RNA-from-Noah's-Heat-Mechanical-Stress-Project/) from Noah's [_Crassostrea gigas_ (Pacific oyster)](http://en.wikipedia.org/wiki/Pacific_oyster) RNA ([isolated 20230712](https://robertslab.github.io/sams-notebook/2023/07/12/RNA-Isolation-C.gigas-Ctenidia-from-Noah's-Heat-Mechanical-Stress-Project/)), 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](#results) section below for cycling params, etc.

qPCR calcs:

- [20230713 - qPCR Calcs Noah gigas ctenidia pooled cDNA](https://docs.google.com/spreadsheets/d/1ie8IrIDFY-KTxb2Iwwyyi-e03WaNRGPxSrYbYsC0kG0/edit?usp=sharing) (Google Sheet)

---

# RESULTS

qPCR Data file (QPCRD - CFX Maestro required)

- [sam_2023-07-13_13-15-17_Connect.pcrd](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-13_13-15-17_Connect.pcrd)

qPCR Report (PDF)

- [sam_2023-07-13_13-15-17_Connect.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2023-07-13_13-15-17_Connect.pdf)

Unsurprisingly, the two primer sets designed for [_Ostrea lurida_ (Olympia oyster)](http://en.wikipedia.org/wiki/Pacific_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.



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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](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-13_13-15-17_Connect-amp_plots.png)

---

Melt Curves

- Defensin in GREEN
- IL-17 in BLACK

![qPCR melt plots for defensin (green) and Il-17 (black).](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2023-07-13_13-15-17_Connect-melt_plots.png)