qPCRs - C.gigas Lifestage Carryover DNMT1 GAPDH HSP70 and HSP90

INTRO

Continuing work with project-gigas-carryover lifestage_carryover (GitHub repo) after creating cDNA on 20250516, it was finally time to run the qPCRs.

This notebook describes how the qPCRs were run and links to the various output files. It also provides a brief overview of each primer set’s amplification profiles. This notebook does not have any analysis. This will be performed later.

MATERIALS & METHODS

Based on results from an initial round of qPCRs on 20240327 (Notebook entry), the following primer sets were run:

SR ID	Primer Name
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
1510	Cg_DNMT1_F
1511	Cg_DNMT1_R

All samples were run in triplicate, on low-profile, white 96-well plates (USA Scientific) in a CFX Connect (Bio-Rad) or CFX96 (Bio-Rad) real-time thermalcycler. All reactions consisted of the following:

Component	Stock Concentration	Volume (uL)
cDNA	NA	1
SsoAdvanced Universal SYBR Green Supermix (BioRad)	2x	10
PF	10uM	0.5
PR	10uM	0.5
H2O	NA	8
TOTAL		20

Master mixes were distributed across one plate for each primer set and included no template controls (NTCs).

For cycling parameters, plate layouts, etc. see the RESULTS section below.

RESULTS

Summary

• GAPDH: Amplification and melt plots only look okay. No template controls appear to have some amplification, however the melt curve of the NTCs is at a different temperature than the other samples. This suggests that the remainder of the samples are fine for analysis. Reps are tight.

  Of note, sample 29 has a standard deviation that is too large and will require repeating the qPCRs.

• HSP70: Amplification and melt plots look good. No amplification in NTCs. Reps are tight.

• HSP90: Amplification and melt plots look good. Reps are tight.

  Of note, samples 14, 18, and 29 have each have a large standard deviation. These will need to be repeated.

• DNMT1: Amplification and melt plots look good. No amplification in NTCs. Reps are tight.

Files

Tip

• *.pdf: qPCR Reports. Contains plate layouts, cycling params, amp/melt plots, etc.

• *Amplification-Results_SYBR.csv: Raw fluorescence data.

• *Cq-Results.csv: Cycle quantity (Cq) data.

• *.pcrd: Source qPCR data file. Requires CFX Maestro (Bio-Rad) software to open.

All files linked below are from commit b089754.

Also, this is all raw data. None of it has been normalized, so cannot interpret results from this data.

GAPDH

Cq Data

• sam_2025-06-09_13-59-10_CFX96-GAPDH-Quantification-Cq-Results.csv

Report

• sam_2025-06-09_13-59-10_CFX96-GAPDH.pdf

Raw fluorescence

• sam_2025-06-09_13-59-10_CFX96-GAPDH-Quantification-Amplification-Results_SYBR.csv

CFX File

• sam_2025-06-09_13-59-10_CFX96-GAPDH.pcrd

HSP70

Cq Data

• sam_2025-06-09_12-56-08_CFX96-HSP70-Quantification-Cq-Results.csv

Report

• sam_2025-06-09_12-56-08_CFX96-HSP70.pdf

Raw fluorescence

• sam_2025-06-09_12-56-08_CFX96-HSP70-Quantification-Amplification-Results_SYBR.csv

CFX File

• sam_2025-06-09_12-56-08_CFX96-HSP70.pcrd

HSP90

Cq Data

• sam_2025-06-09_13-34-41_Connect-HSP90-Quantification-Cq-Results.csv

Report

• sam_2025-06-09_13-34-41_Connect-HSP90.pdf

Raw fluorescence

• sam_2025-06-09_13-34-41_Connect-HSP90-Quantification-Amplification-Results_SYBR.csv

CFX File

• sam_2025-06-09_13-34-41_Connect-HSP90.pcrd

DNMT1

Cq Data

• sam_2025-06-09_12-34-43_Connect-DNMT1-Quantification-Cq-Results.csv

Report

• sam_2025-06-09_12-34-43_Connect-DNMT1.pdf

Raw fluorescence

• sam_2025-06-09_12-34-43_Connect-DNMT1-Quantification-Amplification-Results_SYBR.csv

CFX File

• sam_2025-06-09_12-34-43_Connect-DNMT1.pcrd

Plots

(a) GAPDH Amplification plot

(b) GAPDH Melt plot

Figure 1: GAPDH qPCR plots.

(a) HSP70 Amplification plots

(b) HSP70 Melt plots

Figure 2: HSP70 qPCR plots.

(a) HSP90 Amplification plots

(b) HSP90 Melt plots

Figure 3: HSP90 qPCR plots.

(a) DNMT1 Amplification plots

(b) DNMT1 Melt plots

Figure 4: DNMT1 qPCR plots.