---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: qPCR - P.generosa APLP and TIF3s8-1 with cDNA
date: '2020-08-06 08:21'
tags:
  - qPCR
  - CFX Connect
  - BioRad
  - 2x SsoFast EvaGreen
  - SRID:1768
  - SRID:1769
  - SRID:1758
  - SRID:1759
  - cDNA
categories:
  - 2020
  - Miscellaneous
---
[Shelly asked me to run some qPCRs](https://github.com/RobertsLab/resources/issues/982) (GitHub Issue), after some of the qPCR results I got from primer tests with [normalzing genes](https://robertslab.github.io/sams-notebook/posts/2020/2020-07-29-qPCR-Testing-P.generosa-Reproduction-related-Primers/) and [potential gene targets](https://robertslab.github.io/sams-notebook/2020/07/23/qPCR-Testing-P.generosa-Reproduction-related-Primers/).


Primers used:

| SRID | Primer_Name |
|------|-------------|
| 1769 | APLP_FWD    |
| 1768 | APLP_REV    |
| 1773 | TIF3s8_FWD-1 |
| 1772 | TIF3s8_REV-1 |

- TIF3s8 is expected to be used as a normalizing gene.


The cDNA being used will be samples [made by Kaitlyn on 20200212](https://genefish.wordpress.com/2020/02/12/kaitlyns-notebook-testing-new-primers-on-geoduck-hemolymph-rna/). Samples used are those lined up towards the top of the sample box. It's unclear to me why there are some samples in 0.5mL tubes and others in 1.7mL tubes:

![Pic of Kaitlyn's geoduck samples box](https://github.com/RobertsLab/sams-notebook/blob/master/images/20200806_kaitlyn_geoduck_cDNA_box.jpg?raw=true)


Positive control was pooled cDNA, created by combining 2uL from each of the following:

- 11-08 1H ([made by me from 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-26-Reverse-Transcription---P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/))
- 11-08 2H ([made by me from 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-26-Reverse-Transcription---P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/))
- 57H ([made by me from 20191125](https://robertslab.github.io/sams-notebook/posts/2019/2019-11-26-Reverse-Transcription---P.generosa-DNased-Hemolypmh-and-Hemocyte-RNA-from-20191125/))
- 11/15 Chew (made by Kaitlyn, no date on tube)
- 11/21 Star (made by Kaitlyn, no date on tube)


All qPCR reactions were run in duplicate. See qPCR Report (Results section below) for plate layout, cycling params, etc.


Master mix calcs are here:

- [20200806_qPCR_geoduck_APLP_TIF3s8](https://docs.google.com/spreadsheets/d/1lDXLltnToDF-NVxRfDtGuwAye4gN8D0N6LftgLlwUBQ/edit?usp=sharing) (Google Sheet)

---

# RESULTS

qPCR Report (PDF):

- [sam_2020-08-06_05-44-24_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2020-08-06_05-44-24_BR006896.pdf)

CFX Data File (PCRD):

- [sam_2020-08-06%2005-44-24_BR006896.pcrd](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-06%2005-44-24_BR006896.pcrd)


CFX Results File (CSV):

- [sam_2020-08-06_05-44-24_BR006896-Quantification_Cq_Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-06_05-44-24_BR006896-Quantification_Cq_Results.csv)

---

Plot color legend:

- `APLP`: BLACK

- `Positive control`: GREEN

- No Template Controls: RED


#### APLP Amplification plots

![APLP amplifcation plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-06_05-44-24_BR006896_APLP_amp_plots.png)

#### APLP Melt curves
![APLP melt curves](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-06_05-44-24_BR006896_APLP_melt_plots.png)

---

Plot color legend:

- `TIF3s8-1`: ORANGE

- `Positive control`: GREEN

- No Template Controls: RED

#### TIF3s8-1 Amplification plots

![TIF3s8-1  amplifcation plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-06_05-44-24_BR006896_TIF3s8-1_amp_plots.png)

#### TIF3s8-1  Melt curves
![TIF3s8-1 melt curves](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-06_05-44-24_BR006896_TIF3s8-1_melt_plots.png)

---

Overall, the data itself looks fine. There are a few samples here and there where the replicates aren't great; primarily those that amplify very late (>37 Cq). This isn't terribly unusual, but as a mild perfectionist, it annoys me.

However, upon some brief analysis, it's clear that using `TIF3s8-1` as a normalizing gene will _not_ work. It fails to amplify in the majority of samples. Cross-checking with the results of `APLP` amplification in those same samples shows that `APLP` _did_ amplify in most of those same samples; thus ruling out an issue with the samples themselves.

Will let Shelly know and will probably come up with a plan for identifying new normalizing gene targets.