qPCR - C.gigas Alternative Oxidases Primer Tests

INTRO

I previously designed primers for three alternative oxidase (AOX) variants on 20250117 (Notebook entry). Alternative oxidase is a mitochondrial gene/enzyme known to be responsive to oxidative stress, so this may be a useful biomarker that we haven’t previously considered.

I needed to test the primer sets to see if they worked and were template-specific. I also used this as an opportunity to introduce some of the undergrads in lab to qPCR. Carolyn, Madeline, and Noah all participated.

Alternative oxidase variants

The primers were designed on the following NCBI sequences:

• NP_001292289.1: alternative oxidase, mitochondrial-like Magallana gigas

  • Nucleotide: NM_001305360.1

  • Gene: LOC105322704

• XP_034318738.2: alternative oxidase, mitochondrial-like isoform X2 Magallana gigas

  • Nucleotide: XM_034462847.2

  • Gene: LOC105322704

• XP_065939571.1: alternative oxidase, mitochondrial-like isoform X1 Magallana gigas

  • Nucleotide: XM_066083499.1

  • Gene: LOC105322704

MATERIALS & METHODS

Primers

Gene	SR_ID	Primer Name
AOX vX1	1849	cgig-AOX-XM_066083499.1-R
AOX vX1	1848	cgig-AOX-XM_066083499.1-F
AOX vX2	1847	cgig-AOX-XM_034462847.2-R
AOX vX2	1846	cgig-AOX-XM_034462847.2-F
AOX	1845	cgig-AOX-NM_001305360-R
AOX	1844	cgig-AOX-NM_001305360-F

Note

The vX1 and vX2 are NCBI notations to indicate different isoforms.

Reactions

All samples were run in duplicate, on low-profile, white 96-well plates (USA Scientific) in a CFX Connect (BioRad) real-time thermalcycler. All reactions consisted of the following:

Component	Stock Concentration	Volume (uL)
cDNA	NA	1
SsoAdvanced Universal SYBR Green Supermix (BioRad)	2x	10
PF	10uM	0.5
PR	10uM	0.5
H2O	NA	8
TOTAL		20

A pool of cDNA from the various samples in the lifestage carryover project was used as the source of cDNA, in hopes of obtaining a source of cDNA with AOX1 expression.

For cycling parameters, plate layouts, etc. see the RESULTS section below.

RESULTS

Summary

All three primers work great! Amplfication is seen in all three primers, with AOX (SR IDs 1844/5) coming up the earliest at around 28 Cqs and AOX vX2 (SR IDs 1846/7) coming up the latest at around 33 Cqs. Technically, these values don’t really matter, but they do show that AOX expression is likely at high enough levels to be easily detected in future experiments. No amplification is present in the no template controls (NTCs).

Plots

Figure 1: Amplification plots

Figure 2: Melt plots.

• AOX (SR IDs 1844/5) is shown in gold.
• AOX vX2 (SR IDs 1846/7) is shown in light blue.
• AOX vX1 (SR IDS 1848/9) is shown in dark blue.
• NTCs are shown in red.

Data file

qPCR Data file (CFX Maestro):

• admin_2025-05-27_11-07-38_Connect-cgig-AOX-primers.pcrd

Undergrad results

Undergrads did great! Replicates are tight and results mirror what I obtained. Additionally, despite potential amplification in NTCs (as seen in red in the plots below), this amplification did not occur in both of their replicates and did not occur in any of their samples containing template. Very nice!

Plots

Figure 3: Undergrad Amplification plots

Figure 4: Undergrad Melt plots.

Undergrad qPCR amplification and melt plots. See data file (admin_2025-05-27_11-07-38_Connect-cgig-AOX-primers.pcrd) for which samples belong to each person.