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Primer database

Nkx2-1

NK2 homeobox 1

Rattus norvegicus

PublicationSousa-Vidal et al. 2023

Sequences

Forward (5′→3′)

ACCTTACCAGGACACCATGC

Reverse (5′→3′)

TTCTTGCTCACGTCCCCCAG

Amplicon

162 bp

estimated from mapping

Primer Tm (°C)

ConditionsForwardReverseAverageΔ F/R
qPCR master mix63.666.164.92.5
Primer-BLAST default59.762.160.92.4

qPCR master mix: Owczarzy 2004/2008 · 250 nM primer · 50 mM Na⁺ · 3 mM Mg²⁺ · 0.8 mM dNTP

Primer-BLAST default: SantaLucia 1998 salt corr. · 50 nM primer · 50 mM Na⁺ · 1.5 mM Mg²⁺ · 0.6 mM dNTP

Bold, underlined base = 3′ end (polymerase extension point).

Primer QC

GC% · Forward

55.0%

GC% · Reverse

60.0%

Self-dimer ΔG · Fwd (any position)

-1.9 kcal/mol

Self-dimer ΔG · Fwd (3′ end)

-0.3 kcal/mol

Self-dimer ΔG · Rev (any position)

-3.0 kcal/mol

Self-dimer ΔG · Rev (3′ end)

0.7 kcal/mol

Heterodimer ΔG (F×R, any position)

-2.7 kcal/mol

Heterodimer ΔG (F×R, 3′ end)

-0.3 kcal/mol

ΔG at 37 °C · SantaLucia 1998 NN parameters · < −6 kcal/mol = significant dimer risk · "3′ end" is restricted to duplexes anchored at a primer's own 3′ terminal base (polymerase-extendable); "any position" is the most stable duplex anywhere in the primer. Estimated screening value. For comprehensive dimer analysis, validate with Primer3.

Source

PMID

36714560(Europe PMC)

Citation

Sousa-Vidal et al. 2023

Genomic mapping

Ensembl gene

ENSRNOG00000008644 ↗

NCBI Gene ID

25628

Assembly

GRCr8

Cross-check

✓ENSRNOT00000011453★
✓ENSRNOT00000121637
329bp126bp339bp386bp1.9kb
exon presentprimer binds hereconfirmed amplicongap = exon skipped~bp = boundary varies
How do I read this diagram?

Each row is one transcript, aligned on a shared axis. A gap means that isoform skips this exonic region. Each row's box is drawn at that isoform's own real exon length, which can be narrower than the column (alternative splice site); hover a box for its exact size. A ~amber header bp means isoforms don't all agree on this region's boundary, while a plain header means they do. Navy blocks mark where the primers bind, and green (with a ✓ by the name) means this isoform has a confirmed working amplicon, not just exon overlap. A row with a “‹ exons” / “exons ›” hint instead of any box means that isoform's exons exist but sit outside the current scroll position (a truncated isoform or alternate transcription start site) — click the hint to jump to them. Click a row to load it below.

★ = Ensembl canonical transcript (reference model, not necessarily the most expressed isoform)

Showing: ENSRNOT00000011453 ★

339bp1821bp386bp23.2kb1.9kb3FRPredicted from the amplicon's own GC%+length (von Ahsen et al. 2001, at the same qPCR buffer as the primer Tm above: 50 mM Na⁺ plus 3 mM Mg²⁺ less 0.8 mM dNTP, as a sodium equivalent), not the SantaLucia model used for primer Tm, which isn't valid at this length. A screening estimate for ranking amplicons — that relation was fitted on oligonucleotide duplexes, so applying it at amplicon length carries more error than its published ±1.9 °C, and it is not a melt-peak prediction.162 bp amplicon · predicted amplicon Tm 93.9 °C
Nkx2-1 · ENSRNOT00000011453 (NM_013093) · canonical✓ Exon-junction spanning · gDNA-safe (>1.0kb intron)

Off-target specificity

Specific

No paired forward+reverse off-target amplicon found against nt (whole genome/transcriptome). Screen requires >=2 total mismatches including >=2 within the last 5bp at the 3' end to clear a hit (NCBI Primer-BLAST-style stringency); hits with >=6 total mismatches are dropped as implausible.

Efficiency measurements

Ideal range:Dilution series 1.90–2.10·LinRegPCR 1.70–1.90·unknown method: not scored
No measurements yet for this primer pair.

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