---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: qPCR - P.generosa RPL5 and TIF3s6b v2 and v3 Normalizing Gene Assessment
date: '2020-08-25 09:42'
tags:
  - Panopea generosa
  - geoduck
  - qPCR
  - CFX Connect
  - cDNA
categories:
  - 2020
  - Miscellaneous
---
[After testing out the RPL5 and TIF3s6b v2 and v3 primers yesterday](https://robertslab.github.io/sams-notebook/posts/2020/2020-08-24-qPCR---P.generosa-RPL5-v2-v3-and-TIF3s6b-v2-v3-Primer-Tests/) on pooled cDNA, we determined the primers looked good, so will go forward testing them on a set of _P.generosa_ hemolymph [cDNA made by Kaitlyn on 20200212](https://genefish.wordpress.com/2020/02/12/kaitlyns-notebook-testing-new-primers-on-geoduck-hemolymph-rna/). This will evaluate whether or not these can be utilized as normalizing genes for subsequent gene expression analyses.


Primers used:

| SRID | Primer_Name    |
|------|----------------|
| 1787 | RPL5_v2_FWD    |
| 1786 | RPL5_v2_REV    |
| 1785 | RPL5_v3_FWD    |
| 1784 | RPL5_v3_REV    |
| 1783 | TIF3s6b_v2_FWD |
| 1782 | TIF3s6b_v2_REV |
| 1781 | TIF3s6b_v3_FWD |
| 1780 | TIF3s6b_v3_REV |

I used [geoduck gDNA (162ng/uL; from 20170105)](https://robertslab.github.io/sams-notebook/posts/2017/2017-01-05-dna-isolation-geoduck-gdna-for-illumina-initiated-sequencing-project/) as a positive control.

Master mix calcs are here:

- [200200825_qPCR_geoduck_RPL5-v2-v3_TIF2s6b-v2-v3](https://docs.google.com/spreadsheets/d/17VjL38kjRQkRdMdIsdqPhtWCFAvkX_dJTY-DX2iCLTA/edit?usp=sharing) (Google Sheet)

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

NOTE: These qPCRs used the remainder of all the samples.

---

# RESULTS


These primers are not going to be useful as normalizing genes:

- RPL5 spread is way too wide for use as a normalizing gene (~10 Cqs)

- TIF3s6b doesn't amplify in most samples (however, it is very consistent in those that it does amplify...)

Data files and amplification/melt plots are below.

RPL5 qPCR Report (PDF):

- [sam_2020-08-25_05-22-37_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2020-08-25_05-22-37_BR006896.pdf)

RPL5 CFX Data File (PCRD):

- [sam_2020-08-25%2005-22-37_BR006896.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2020-08-25%2005-22-37_BR006896.pcrd)

RPL5 CFX Results File (CSV):

- [sam_2020-08-25_05-22-37_BR006896-Quantification-Cq-Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_05-22-37_BR006896-Quantification-Cq-Results.csv)


TIF3s6b qPCR Report (PDF):

- [sam_2020-08-25_06-59-18_BR006896.pdf](https://owl.fish.washington.edu/Athaliana/qPCR_data/qPCR_reports/sam_2020-08-25_06-59-18_BR006896.pdf)

TIF3s6b CFX Data File (PCRD):

- [sam_2020-08-25%2006-59-18_BR006896.pcrd](https://owl.fish.washington.edu/scaphapoda/qPCR_data/cfx_connect_data/sam_2020-08-25%2006-59-18_BR006896.pcrd)

TIF3s6b CFX Results File (CSV):

- [sam_2020-08-25_06-59-18_BR006896-Quantification-Cq-Results.csv](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_06-59-18_BR006896-Quantification-Cq-Results.csv)


##### RPL5 v2



AMPLIFICATION PLOTS

![RPL5 v2 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_05-22-37_BR006896_RPL5-v2_amp-plots.png)

MELT PLOTS

![RPL5 v2 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_05-22-37_BR006896_RPL5-v2_melt-plots.png)

##### RPL5 v3

![RPL5 v3 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_05-22-37_BR006896_RPL5-v3_amp-plots.png)

![RPL5 v3 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_05-22-37_BR006896_RPL5-v3_melt-plots.png)

##### TIF3s6b v2

![TIF3s6b v2 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_06-59-18_BR006896_TIF3s6b-v2_amp-plots.png)

![TIF3s6b v2 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_06-59-18_BR006896_TIF3s6b-v2_melt-plots.png)


##### TIF3s6b v3

![TIF3s6b v3 amplification plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_06-59-18_BR006896_TIF3s6b-v3_amp-plots.png)

![TIF3s6b v3 melt plots](https://owl.fish.washington.edu/Athaliana/qPCR_data/sam_2020-08-25_06-59-18_BR006896_TIF3s6b-v3_melt-plots.png)
