INTRO
Continuing work with project-gigas-carryover lifestage_carryover (GitHub repo) after creating cDNA on 20241024, it was finally time to run the qPCRs. Three primer sets were run yesterday, 20241210: GAPDH, ATP synthase, and cGAS.
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.
Due to unequal quantities of RNA used to make cDNA, information from Cq values will not provide meaningful information without normalization first.
MATERIALS & METHODS
Based on results from an initial round of qPCRs on 20240327 (Notebook entry), the following primer sets were selected:
| SR ID | Primer Name |
|---|---|
| 1383 | Cg_citratesynthase_F |
| 1384 | Cg_citratesynthase_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 2.5 plates for each primer set. No template controls (NTC) were loaded on on just one of the three plates (plate #3 in all instances).
For cycling parameters, plate layouts, etc. see the RESULTS section below.
RESULTS
Summary
Citrate synthase: Amplification and melt plots look good. Of note, amplification in243is noticeably later than all other samples. No amplification in NTCs. Reps are tight.DNMT1: Amplification and melt plots look good. No amplification in NTCs.HSP70: Amplification and melt plots look good. No amplification in NTCs. Sample244has a terrible set of reps (std. dev. of 4!!). All other reps are tight.HSP90: Amplification and melt plots look good. No amplification in NTCs.
Files
*.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 e816421.
Citrate synthase
Cq Data
Reports
Raw fluorescence
CFX Files
DNMT1
Cq Data
Reports
Raw fluorescence
CFX Files
HSP70
Cq Data
Reports
Raw fluorescence
CFX Files
HSP90
Cq Data
Reports
Raw fluorescence
CFX Files
Plots
