qPCR - P.generosa RPL5 and TIF3s6b v2 and v3 Normalizing Gene Assessment

After testing out the RPL5 and TIF3s6b v2 and v3 primers yesterday 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. 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) as a positive control.

Master mix calcs are here:

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):

RPL5 CFX Data File (PCRD):

RPL5 CFX Results File (CSV):

TIF3s6b qPCR Report (PDF):

TIF3s6b CFX Data File (PCRD):

TIF3s6b CFX Results File (CSV):

RPL5 v2

AMPLIFICATION PLOTS

RPL5 v2 amplification plots

MELT PLOTS

RPL5 v2 melt plots

RPL5 v3

RPL5 v3 amplification plots

RPL5 v3 melt plots

TIF3s6b v2

TIF3s6b v2 amplification plots

TIF3s6b v2 melt plots

TIF3s6b v3

TIF3s6b v3 amplification plots

TIF3s6b v3 melt plots