qPCR - P.generosa APLP and TIF3s8-1 with cDNA

Shelly asked me to run some qPCRs (GitHub Issue), after some of the qPCR results I got from primer tests with normalzing genes and potential gene targets.

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. 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

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

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

Master mix calcs are here:


RESULTS

qPCR Report (PDF):

CFX Data File (PCRD):

CFX Results File (CSV):


Plot color legend:

  • APLP: BLACK

  • Positive control: GREEN

  • No Template Controls: RED

APLP Amplification plots

APLP amplifcation plots

APLP Melt curves

APLP melt curves


Plot color legend:

  • TIF3s8-1: ORANGE

  • Positive control: GREEN

  • No Template Controls: RED

TIF3s8-1 Amplification plots

TIF3s8-1  amplifcation plots

TIF3s8-1 Melt curves

TIF3s8-1 melt curves


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.