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SameSpots has a simple workflow that allows quick and objective differential expression analysis. Alignment removes positional variation between gels and allows a single spot pattern to be detected, allowing multiple experimental designs to be analysed.

SpotCheck is a workflow within SameSpots that allows objective validation of gel running within the lab.

SameSpots has a simple linear workflow which is quick and reproducible.

Simple workflow of SameSpots for differential expression

Images – Alignment – Spot Detection – Automatic Analysis – Statistics – Reporting


Images are automatically quality checked when uploaded to the software, giving you instant feedback before you start analysis. In the case of analysing a DIGE or multi-stained gel experiment, images are grouped according to the gel.

An alignment reference image is then selected and any areas of the images to be excluded from the analysis, e.g. damage, air bubbles, or edges of the gel, are masked from the analysis.


Image QC highlights any potential issues which you should be aware of prior to analysis.


Alignment removes any positional variation between the spot patterns of the gels introduced during the electrophoresis and imaging processes. Alignment is performed at the pixel level to provide direct and accurate comparison of images allowing a single spot pattern to be detected.

Alignment is often completed using vectors which are automatically created, manual vectors can be added to aid the alignment.

Alignment removes positional variation and aids 100% spot matching across gels.

Spot Detection

Spots are automatically co-detected on all images in the analysis, creating a single spot pattern of the analysis. Normalised spot volumes are automatically calculated following detection.

The filtering stage allows removal of spots based upon position, area, normalised volume or a combination of these. Normalised spot volumes are automatically recalculated following filtering.

Automated co-detection of spots on all gel images in the analysis.

Experimental Set Up and Automatic Analysis

Images can be analysed by three experimental designs:

  • Between subject – samples from a single subject appear only in one condition.
  • Within subject – Samples from a given subject were taken under different conditions, e.g. a time series study
  • Two-way ANOVA – Samples from a given subject appear in two conditions.

Images are grouped into conditions to be analysed and multiple experimental designs can be set up.

ANOVA p-values and fold change between each of the conditions are automatically calculated. Each spot can be visualised in a number of ways with expression profiles and spot details available.

Spots can be sorted by p-value or fold change, spots of interest can be tagged and the list can be filtered to include only the spots of interest. Additional details can be added to the spots including MS data, pI and MW information or notes.


Three experimental set ups allow you to review spots in the most appropriate way.

Statistical Analysis

SameSpots has two major advantages for statistical analysis of spots due to alignment of the images followed by detection of a single spot pattern:

  1. No missing values.
  2. Consistent spot outlines.

Statistical analyses which can be completed within SameSpots include:

  • Principle component analysis (PCA),
  • Correlation analysis – dendrogram,
  • ANOVA p-value calculation,
  • Power analysis and
  • q-value calculation.
Comprehensive multivariate statistical analysis is available within SameSpots

Spot Picking and Reporting

Spot picking files can be created for use with most of the common picking robots and information for manual picking can be exported.

A HTML report of results can be created from selected fields.

Clip Gallery is a tool which allows any images, tables or graphs from the analysis to be exported in high quality for use in reports and presentations.

A simple HTML report can be created and shared in seconds.

Further Reading - Workflow Guide

Contact us to try SameSpots on your own images today: