qPCR - P.generosa RPL5-v2-v3 and TIF3s6b-v2-v3 Primer Tests

Shelly ordered some new primers as potential normalizing genes and asked me to test them out (GitHub Issue).

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

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

I also used geoduck gDNA (162ng/uL; from 20170105) as a potential positive control, and/or as confirmation that these primers will/not amplify gDNA.

Master mix calcs are here:

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


RESULTS

qPCR Report (PDF):

CFX Data File (PCRD):

CFX Results File (CSV):

All the primers look good:

  • Cq is reasonably low

  • Melt curves have single peak

Amplifcation/melt plots for each primer are below.

NOTE: Genomic DNA is amplified by all three primer sets and comes up at an earlier Cq than cDNA. In TIF3s6b v3, gDNA exhibits a small, secondary peak at ~75oC.

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