Target selection
Search NCBI RefSeq transcripts by gene and organism, or provide a DNA sequence directly. Transcript choice defines the template and, for RT-qPCR, the exon structure used downstream.
RefSeq · PCR · RT-qPCR · Primer3
Molecular Assay Design Workflow
Move from a biological target to a defensible PCR or RT-qPCR assay plan. Search NCBI RefSeq transcripts, choose the assay type, define primer-design constraints, generate ranked candidates and review specificity and RT-qPCR-specific risks before validation.
NCBI RefSeq target selection
Standard PCR & RT-qPCR
Primer3 candidate generation
Specificity review
NCBI RefSeq Target Selection
Enter a gene symbol, choose Human or Mouse, search NCBI RefSeq transcripts, then load the selected transcript/cDNA and its corresponding genomic DNA into separate boxes below.
Find another organism in NCBI Taxonomy
Human and Mouse remain available above. Use this search only when you need another NCBI organism.
Search NCBI Taxonomy, then choose the correct organism.
Enter a gene symbol and search RefSeq.
No transcript selected yet.
Research use only. RefSeq retrieval provides sequence context; transcript choice should match the biological question.
Genomic DNA / FASTA Template for Primer Design
Gene-oriented genomic DNA loads separately from cDNA when a RefSeq transcript is selected. You may also paste a shorter DNA target region or explicitly use the transcript as your primer template.
Select a RefSeq transcript to retrieve its corresponding genomic DNA, or paste your own DNA/FASTA target region. The cDNA button below is an explicit alternative.
Select a genomic target region
Keep the full genomic DNA above. Choose a small, meaningful region for Primer Design; coordinates are 1-based positions in the displayed genomic DNA.
Recommended 500–2,000 nt; maximum 5,000 nt for online design.
Primer target region
Full genomic genes can be very large. Keep the complete genomic DNA above,
but send only a defined target region to Primer Design.
Coordinates are 1-based positions within the loaded genomic sequence.
Recommended for long genomic genes: choose a focused region around the experimental target.
FASTA headers, spaces and line numbers are ignored. U is converted to T.
Unsupported sequence letters are reported rather than silently removed.
Protein Product
Protein product annotated for the selected RefSeq transcript CDS.
Load a RefSeq transcript above to retrieve its annotated protein product.
Sequence relationship:
transcript/cDNA is used for primer and RT-qPCR design; the transcript-linked
CDS translation is used for peptide and MHC/HLA workflows.
RT-qPCR + Hydrolysis Probe Design
Use the loaded RefSeq transcript above to generate exon-aware RT-qPCR primer pairs and ranked internal hydrolysis-probe candidates.
Load a RefSeq transcript above. Exon coordinates will be carried into the RT-qPCR design.
Waiting for a transcript or DNA sequence.
Probe candidates are sequence-based computational suggestions. Fluorophore/quencher chemistry, amplification efficiency, specificity and assay performance require experimental validation.
Assay planning
From target to candidateApplication choice
Standard PCR emphasizes robust primer-pair generation and specificity review. RT-qPCR adds exon-aware interpretation, genomic-DNA risk assessment and quantitative-assay considerations.
Parameter control
Tm, GC content and product-size constraints define which primer pairs can be returned. Narrower windows produce more uniform candidates; wider windows increase design flexibility.
Six-Step Molecular Assay Design Workflow
1 · TargetChoose a RefSeq transcript or a defined DNA template.
2 · ApplicationSelect Standard PCR or RT-qPCR according to the experimental objective.
3 · ConstraintsSet Tm, GC and amplicon-size windows plus optional background sequences.
4 · GenerateUse Primer3-based design to create several candidate primer pairs.
5 · ReviewCompare structure, specificity and RT-qPCR-specific annotations.
6 · ValidateConfirm the final assay experimentally with appropriate controls.
Standard PCR vs RT-qPCR
| Design consideration | Standard PCR | RT-qPCR |
|---|---|---|
| Primary purpose | Specific amplification of a defined DNA region | Quantitative measurement of cDNA derived from RNA |
| Template annotation | Useful but not always essential | Important when exon-aware placement or gDNA-risk assessment is needed |
| Amplicon strategy | Size depends on downstream use | Shorter amplicons are generally favored for efficient quantitative cycling |
| Genomic DNA | Depends on the experimental template | Can confound expression measurements and should be controlled |
| Additional review | Primer structure and specificity | Primer structure, specificity, exon placement, gDNA risk and assay efficiency |
Scientific diagrams
RT-qPCR and specificity logicWhy RefSeq Transcript Choice Matters
A gene can have multiple transcript isoforms with different exon structures. If the biological question is isoform-specific, primer placement must be chosen accordingly. If the goal is total-gene expression, a shared region may be more appropriate. For RT-qPCR, transcript annotation also determines which exon boundaries are available for exon-aware designs.
Practical interpretation: transcript selection is a biological decision, not just a sequence-retrieval step.
The same gene symbol can lead to different assays depending on which transcript is chosen.
Research use: review candidate specificity and validate the final assay experimentally.