qPCR - Geoduck Normalizing Gene Primer Checks

Shelly ordered some new primers (designed by Sam Gurr) (GitHub Issue) to potentially use as normalizing genes for her geoduck reproduction gene expression project and asked that I test them out.

Primers tested:

SRID	Primer_Name
1803	28s_v1_FWD
1802	28s_v1_REV
1801	28s_v2_FWD
1800	28s_v2_REV
1799	28s_v3_FWD
1798	28s_v3_REV
1797	28s_v4_FWD
1796	28s_v4_REV
1795	EF1a_v1_FWD
1794	EF1a_v1_REV
1793	EF1a_v2_FWD
1792	EF1a_v2_REV
1791	EF1a_v3_FWD
1790	EF1a_v3_REV
1789	EF1a_v4_FWD
1788	EF1a_v4_REV

I used pooled cDNA, created by combining 2uL from a variety of cDNA previously made by Kaitlyn (sorry, didn’t document which samples contributed this time…).

I also used a 1:10 dilution of 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:

200200914_qPCR_geoduck_28s-v1-4_EF1a-v1-4 (Google Sheet)

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

RESULTS

qPCR Report (PDF):

sam_2020-09-14_10-15-48_BR006896.pdf

CFX Data File (PCRD):

sam_2020-09-14_10-15-48_BR006896.pcrd

CFX Results File (CSV):

sam_2020-09-14_10-15-48_BR006896_Quantification-Cq-Results.csv

Overall, all primer sets amplified gDNA and cDNA. All melt curves look really good, although for 28s I’d lean towards using 28s v4 due to the fact that the melt curve doesn’t mirror the gDNA melt curve like the others do. For the same reasons, I’d lean towards using EF1a v1.

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