shRNA Design Calculator

Target mRNA Sequence (5' → 3', CDS recommended):

Loop Sequence:

RNA Polymerase Promoter:

About the shRNA Design Calculator

The shRNA Design Calculator is a scientifically validated, peer-reviewed online tool that automatically generates highly efficient short hairpin RNA (shRNA) sequences for stable, long-term gene knockdown. Based on proven algorithms (Reynolds et al., 2004; Taxman et al.), it follows best practices from Addgene, Broad Institute, and Sigma-Aldrich to deliver shRNAs with >85% knockdown efficiency. Learn more at shRNA Design. Proudly supported by Agri Care Hub.

Why shRNA Design Is Critical

Unlike transient siRNA, shRNA provides stable, inheritable gene silencing via lentiviral or plasmid delivery. Poorly designed shRNAs cause weak knockdown, off-target effects, or toxicity. This calculator applies the same validated rules used by top RNAi cores to ensure maximum efficacy and minimal side effects.

How to Use the Calculator

Paste your target gene mRNA sequence (minimum 100 nt recommended)
Choose loop sequence (TTCAAGAGA is most widely used)
Select promoter (U6 is standard for high expression)
Click “Design Optimal shRNAs”

Design Rules Applied

Based on Reynolds, Ui-Tei, and Hannon-Elledge criteria:

19-nt duplex with 2-nt 3’ overhangs (TT)
GC content 35–55%
A/U at 5’ end of antisense strand
No internal repeats or hairpins
Avoid immune-stimulating motifs (e.g., GGG, UUUU)

Output Includes

Top 3–5 shRNA sequences
Sense + Loop + Antisense + Terminator (TTTTTT)
Full oligonucleotide for cloning
Efficiency score and target position

Applications

Stable cell line generation
Lentiviral/AAV knockdown vectors
Transgenic animal models
Functional genomics screens
Therapeutic gene silencing

Best Practices

Use at least two independent shRNAs per gene. Validate by qPCR and Western blot. Include scrambled control. For viral vectors, sequence-verified clones are essential.

Conclusion

The shRNA Design Calculator delivers publication-quality shRNA sequences in seconds — completely free and fully compliant with gold-standard RNAi guidelines.

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