Using cDNA from 20230713, performed qPCR on the following primer sets selected by Steven:

SRID	Primer-name
1408	Cg_18s_F
1409	Cg_18s_R
1385	Cg_ATPsynthase_F
1386	Cg_ATPsynthase_R
1383	Cg_citratesynthase_F
1384	Cg_citratesynthase_R
1172	Cg_GAPDH_205_F
1173	Cg_GAPDH_355_R
599	Cg_HSP70_F
598	Cg_HSP70_R
1532	Cg_Hsp90_F
1533	Cg_Hsp90_R
1829	Cg_VIPERIN_F
1828	Cg_VIPERIN_R

qPCR calcs:

20230719 - qPCR Calcs Noah gigas ctenidia cDNA (Google Sheet)

Reactions were run on white, low-profile, 96-well qPCR plates (USA Scientific). A total of two plates were run. One was run on a CFC Connect (BioRad) and the other on a CFX96 (BioRad). See the qPCR Report in the RESULTS section below for cycling params, plate layouts, etc.

All samples were run in singular. Normally duplicates/triplicates are run. However, due to time and sample constraints for Noah’s project/poster, samples were just run in singular. No template controls were run in duplicate.

RESULTS

Here’s a brief summary of the qPCR data, based on looking at the amplification and melt curve plots below.

18s: No Template Controls (NTCs) show contamination in melt curve and will need to be rerun.

ATP_synthase: Melt curve looks good. Amplification looks good.

Citrate_synthase: Melt curve looks good. Amplification looks good.

GAPDH: NTC’s do show some very late amplification, but does not cross Cq threshold. Amplification plots show distinct separation between groups of samples, suggesting some response to treatments. Needs to be explored further.

HSP70: Amplification looks good. Melt curves are noticeably more broad than other primer sets. Additional scrutiny reveals a potential additional peak. Both aspects suggest that more than one product is being amplified. This is not terribly surprising, as there are numerous HSP70 present in organisms and they share a great deal of sequence homology to one another. Without careful primer design, it’s very easy to end up amplifying more than one gene in the HSP70 family.

HSP90: Melt curve looks good. Amplification looks good.

VIPERIN: Melt curve looks good. Amplification looks good.

qPCR Results file (CSV)