INTRO

Tested 13 new Manila clam (R.philippinarum) primer sets to see if they amplify and if they are template-specific. Primer sets target the following genes:

C1qL3
eIF4A3
HPRT
HSF1
IAP
RPS14
Viperin

cDNA was the pooled gill cDNA I made earlier today (20261028).

Note

No analysis was performed. This was only to assess primer success/specificity.

MATERIALS & METHODS

Primers

SR_ID	PrimerName
1875	Rp-HSF1-827
1874	Rp-HSF1-927
1873	Rp-HSF1-164
1872	Rp-HSF1-291
1871	Rp-Viperin-5068
1870	Rp-Viperin-5197
1869	Rp-Viperin-929
1868	Rp-Viperin-1050
1867	Rp-IAP-1424
1866	Rp-IAP-1559
1865	Rp-IAP-45
1864	Rp-IAP-195
1863	Rp-C1qL3-518
1862	Rp-C1qL3-660
1861	Rp-C1qL3-206
1860	Rp-C1qL3-325
1859	Rp-eIF4A3-2653
1858	Rp-eIF4A3-2830
1857	Rp-eIF4A3-1144
1856	Rp-eIF4A3-1320
1855	Rp-RPS14-406
1854	Rp-RPS14-527
1853	Rp-RPS14-11
1852	Rp-RPS14-186
1851	Rp-HPRT-608
1850	Rp-HPRT-728

Reactions

All samples and no template controls (NTCs) were run in triplicate, on low-profile, white 96-well plates (USA Scientific) sealed with TemPlate TR Select Optical Film (USA Scientific) in a CFX Connect (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
P_F	10uM	0.5
P_R	10uM	0.5
H₂O	NA	8
TOTAL		20

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

Important

Analysis Mode... was set to Target so that the auto-baseline threshold was set for each primer set independently.

RESULTS

Summary

Of the 13 primer sets, 9 produced amplification and 4 produced none. Every primer set that amplified produced a single melt peak in the template wells.

SR IDs	Gene	Mean Cq	Cq SD	NTCs	Result
1850/1	HPRT	N/A	N/A	Clean	No amplification
1852/3	RPS14	35.61	0.282	Clean	Late amplification
1854/5	RPS14	18.61	0.077	Amplified (Cq 19.12); primer dimer	Works, but primer dimer
1856/7	eIF4A3	29.72	0.160	Clean	Works
1858/9	eIF4A3	27.65	0.470	Clean	Works
1860/1	C1qL3	N/A	N/A	Clean	No amplification
1862/3	C1qL3	N/A	N/A	Clean (no Cq)	No amplification
1864/5	IAP	N/A	N/A	Clean (no Cq)	No amplification
1866/7	IAP	32.17	0.305	Clean	Works
1868/9	Viperin	35.77	0.715	Clean	Late amplification
1870/1	Viperin	27.71	0.111	Clean	Works
1872/3	HSF1	34.81	0.369	Clean	Late amplification
1874/5	HSF1	29.69	0.101	Clean	Works

Working primer sets (good amplification, single melt peak, no NTC amplification):

eIF4A3: SR IDs 1856/7, 1858/9
IAP: SR IDs 1866/7
Viperin: SR IDs 1870/1
HSF1: SR IDs 1874/5

Notes:

RPS14 (SR IDs 1854/5): Amplifies early (Cq ~18.6), which is expected for a ribosomal protein gene. The NTCs also amplified (Cq ~19.1), but their melt peak is ~69^oC, compared to ~83^oC in the template wells. So the NTC signal is primer dimer, not contamination. The template wells show only the ~83^oC peak, but NTC Cqs this close to the template Cqs make this primer set questionable for quantification.
Late amplifiers (SR IDs 1852/3, 1868/9, 1872/3): Cqs of ~35-36, each with a single melt peak and clean NTCs. These amplify, but they’re not useful when there’s a better primer set for the same gene. RPS14 SR IDs 1852/3 coming up at Cq ~35.6 (vs. ~18.6 for the other RPS14 set) suggests these primers are pretty inefficient. SR IDs 1868/9 were flagged by CFX Maestro for replicate SD > 0.5.
No amplification (SR IDs 1850/1, 1860/1, 1862/3, 1864/5): HPRT (only one set tested) and both C1qL3 sets failed. One NTC in each of SR IDs 1862/3 and 1864/5 shows a small melt peak, but no Cq was called for either.

By gene:

Gene	Status
eIF4A3	Two working sets
HSF1	One working set (1874/5)
IAP	One working set (1866/7)
Viperin	One working set (1870/1)
RPS14	One set amplifies, but with primer dimer in NTCs (1854/5)
HPRT	No working set
C1qL3	No working set

Files

Tip

*.pdf: qPCR report. Contains plate layout, cycling params, Cq values, QC flags.
*.pcrd: Source qPCR data file. Requires CFX Maestro (Bio-Rad) software to open.