In Silico Northern Blot

Pre-wet-lab small RNA analysis

Screen small RNA libraries against a DNA probe.

Upload collapsed FASTA samples and a 15–29 nt canonical DNA probe. In Silico Northern Blot uses BLAST+ to nominate candidate RNA, then evaluates complete probe-length windows with IUPAC-aware Hamming distance.

Use the result to plan experiments, not as a prediction of hybridization conditions or physical PAGE migration. Wet-lab confirmation is required.

Result availability: private result links and analysis data remain available for up to 90 days.

Run analysis Upload collapsed FASTA samples, enter a DNA probe, and set count and length filters. Check result Open a private result by UUID and explore thresholded matches, tables, and the illustrative blot. Open .isnb Inspect a saved result locally in your browser; the archive is never uploaded.

Supported workflow

A versioned workflow with two matching stages

Standard ambiguous IUPAC RNA remains in the abundance calculation and matching workflow. Characters outside the supported alphabet stop validation.

  1. Prepare each sample

    Sequences are uppercased, T is converted to U, duplicate RNA is merged, the minimum-count cutoff is applied, and CPM is calculated before matching.

  2. Nominate candidates

    The approved BLAST+ preset searches RNA within the selected length range. BLAST is a heuristic prefilter; its scores do not determine final classification.

  3. Classify each candidate

    Every complete probe-length window in each candidate is evaluated with IUPAC-aware lower and upper Hamming bounds across cumulative mismatch thresholds 0–6.

Result interpretation

Guaranteed signal stays separate from ambiguity

Ambiguous bases remain visible without being treated as certain matches.

Guaranteed

The conservative upper bound is within the selected threshold. The RNA contributes its CPM once and can form a band.

Possible due to ambiguous bases

The lower bound is within the threshold but the upper bound is not. The RNA is reported separately and never contributes signal or creates a band.

Diagnostic

Candidates above the threshold, RNA without a BLAST nomination, and RNA shorter than the probe remain available for interpretation. A missing BLAST candidate is not proof that Hamming distance exceeds 6.

Evidence and scope

Traceable outputs and V1 boundaries

Trace the result

Review per-sample validation, the CPM denominator, threshold summaries, selected windows, IUPAC positions, BLAST HSP provenance, and band components.

A versioned .isnb archive preserves parameter snapshots, result tables, previews, and checksums. The local viewer opens it without uploading the file.

Know the limits

V1 evaluates substitutions in complete probe-length windows. It does not model indels, bulges, loops, duplex structure or accessibility, Tm or ΔG, probe chemistry, hybridization or wash conditions, or a specific PAGE system.

The blot is an illustrative view of guaranteed CPM by RNA length, not a simulated gel or absolute signal prediction.