INTRO

Ran qPCRs on the cDNA I made last week (Notebook), as part of the project-gigas-carryover (GitHub Repo). This file provides metadata for the samples being qPCR’d:

20240314_rna_extractions.csv (GitHub; commit: 82f1a4b).

Note

Not all of the samples listed in the sheet above were used in the qPCRs, due to lack of RNA during isolation (Notebook).

The following primer sets were selected, based on previous usage in other stress (heat) experiments:

SR ID	Primer Name
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
1825	Cg_cGAS_F
1824	Cg_cGAS_R
1510	Cg_DNMT1_F
1511	Cg_DNMT1_R

MATERIALS & METHODS

All samples were run in duplicate, on low-profile, white 96-well plates (USA Scientific) in a CFX Connect (BioRad) 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
P_F	10uM	0.5
P_R	10uM	0.5
H₂O	NA	8
TOTAL		20

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

Baseline threshold analysis was set to “Target” (“Fluorophore” is the default) in the CFX Maestro (BioRad) software for each plate.

RESULTS

Summary

ATP Synthase: Amplification and melt plots look good. No amplification in NTCs. Reps are tight.
HSP70: Amplification and melt plots look good. No amplification in NTCs. Sample 244 has a terrible set of reps (std. dev. of 4!!). All other reps are tight.
GAPDH: Amplification and melt plots look good. Range of ~2.00 Cqs is decent and probably permits usage as normalizing gene for this experiment. Late amplification in NTCs, but melt temp peak is noticeably lower than other amplicons. As such, this is not concerning.
cGAS: Amplification and melt plots look good. Late amplification in NTCs, but melt temp peak is ~2^oC lower than other amplicons… Might be some non-specific amplification? Reps are tight. Reps are tight.
HSP90: Amplification and melt plots look good. No amplification in NTCs. Sample 223 reps are poor, but remainder are tight.
VIPERIN: Amplification and melt plots look good. Of note, amplification in 243 is noticeably later than all other samples. No amplification in NTCs. Most reps are tight, but samples 223 and 243 could be better…
Citrate Synthase: Amplification and melt plots look good. Of note, amplification in 243 is noticeably later than all other samples. No amplification in NTCs. Reps are tight.
DNMT1: Amplification and melt plots look good. No amplification in NTCs. Most reps are tight, but 296, 298, 223, 243, and 285 aren’t great…

Files

:{::: {.callout-tip} - *.pdf: qPCR Reports. Contains plate layouts, cycling params, amp/melt plots, etc.

*Amplificaiton_Results*.csv: Raw fluorescence data.
*Cq_Results.csv: Cycle quantity (Cq) data.
*.pcrd: Source qPCR data file. Requires CFX Maestro (BioRad) software to open.

:::}