qPCR - Geoduck Normalizing Gene Primers 28s-v4 and EF1a-v1 Tests

On Monday (20200914), I checked a set of 28s and EF1a primer sets and determined that 28s-v4 and EF1a-v1 were probably the best of the bunch, although they all looked great. So, I needed to test these out on some individual cDNA samples to see if they might be useful as normalizing genes - should have consistent Cq values across all samples/treatments.

Primers tested:

SRID Primer_Name
1797 28s_v4_FWD
1796 28s_v4_REV
1795 EF1a_v1_FWD
1794 EF1a_v1_REV

I tested them on a set of P.generosa hemolymph cDNA made by Kaitlyn on 20200212.

I also used a 1:10 dilution of geoduck gDNA (162ng/uL; from 20170105) as a positive control, as gDNA was amplified by all the primer sets on Monday.

Master mix calcs are here:

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


qPCR Report (PDF):

CFX Data File (PCRD):

CFX Results File (CSV):

28s-v4 melt plots aren’t great and might even possibly have a slight shoulder, suggesting a secondary product. Not great. Additionally, the samples have a fairly large Cq range; not good for a normalizing gene.

EF1a-v1 melt plots look great, but the amplification also exhibits a Cq range that’s too large for a normalizing gene.

With all of that said, I’m starting to think it would be best to re-quant the source RNA and remake cDNA.

Amplifcation and melt plots for each primer set are below. Color coding:

  • RED: No Template Control (NTC)



28s v4


28s-v4 amp plots.png


28s-v4 melt plots.png

EF1a v1


EF1a-v1 amp plots.png


EF1a-v1 melt plots.png