How to Choose MSI Reference Standards for PCR-CE and NGS Assay Validation

For laboratories developing or validating microsatellite instability testing, choosing the right MSI reference standard is a critical step. MSI testing is not only about detecting instability. It is also about proving that the assay can consistently distinguish MSI-H samples from MSS samples under real testing conditions.

This is especially important for laboratories using PCR-CE or NGS-based workflows. Both methods require reliable control materials to evaluate assay performance, monitor workflow consistency and support confidence in MSI classification.

A suitable MSI reference standard should not only have a known MSI status. It should also match the intended application, sample type, analytical platform and validation purpose.

This article explains what laboratories should consider when selecting MSI reference standards for PCR-CE and NGS assay validation.

Why MSI Reference Standards Matter in Assay Validation

MSI assay validation requires more than a theoretical testing protocol. Laboratories need characterized materials that can challenge the assay and confirm whether the workflow performs as expected.

A high-quality MSI reference standard helps answer several practical questions:

• Can the assay correctly identify MSI-H samples?

• Can the assay correctly identify MSS samples?

• Are the results reproducible across different runs?

• Does the workflow perform consistently across different operators?

• Can the platform detect MSI status using the selected marker panel or sequencing design?

• Does the bioinformatics pipeline correctly classify known MSI-H and MSS samples?

Without appropriate reference standards, it is difficult to evaluate whether unexpected results are caused by the sample, the assay design, the instrument, the reagent lot or the data analysis process.

Key Features to Look for in an MSI Reference Standard

When selecting MSI reference materials, laboratories should evaluate several important features.

1. Clearly Confirmed MSI Status

The first requirement is a clearly defined MSI status.

For MSI assay validation, laboratories usually need both positive and negative reference materials:

• MSI-H reference standards as positive controls

• MSS reference standards as negative or stable controls

MSI-H materials help confirm that the assay can detect microsatellite instability. MSS materials help verify that the assay does not incorrectly classify stable samples as unstable.

For a complete validation workflow, using only MSI-H materials is not enough. Laboratories also need MSS materials to evaluate specificity and classification accuracy.

2. Availability of Paired Tumor and Normal gDNA

Paired tumor-normal genomic DNA is highly useful for MSI testing because many MSI workflows compare tumor DNA against normal DNA.

In PCR-CE workflows, paired normal DNA can provide a baseline for fragment size comparison. This helps laboratories identify tumor-specific shifts in microsatellite markers.

In NGS workflows, paired tumor-normal materials can support workflow development, tumor-normal comparison strategies and pipeline verification.

For laboratories that want a more realistic validation model, paired tumor and normal gDNA is more informative than single unpaired control materials.

3. Compatibility with PCR-CE Workflows

PCR-CE remains a widely used method for MSI detection. It relies on PCR amplification of selected microsatellite markers followed by capillary electrophoresis-based fragment analysis.

When choosing MSI reference standards for PCR-CE, laboratories should consider whether the material supports the markers used in their assay.

Common MSI markers include:

• BAT-25

• BAT-26

• MONO-27

• NR-21

• NR-24

• NR-27

Reference standards with known performance across these markers can help laboratories verify amplification, fragment sizing, marker instability and MSI classification.

For PCR-CE workflows, suitable reference standards can be used to support:

• Marker panel verification

• Fragment analysis consistency

• MSI-H and MSS classification

• Inter-run reproducibility

• Operator training

• Routine positive and negative control testing

4. Compatibility with NGS-Based MSI Detection

NGS-based MSI testing is becoming increasingly common because laboratories can integrate MSI detection into broader cancer genomic profiling workflows.

However, NGS-based MSI detection depends on several technical factors, including:

• Panel design

• Sequencing depth

• Microsatellite locus coverage

• DNA quality

• Tumor fraction

• Library preparation

• MSI calling algorithm

• Bioinformatics threshold settings

Because of this complexity, NGS workflows require carefully selected reference materials.

MSI reference standards can help laboratories evaluate whether the full NGS workflow, from DNA input to final MSI calling, can correctly classify known MSI-H and MSS samples.

For NGS assay validation, reference standards can support:

• Targeted panel validation

• Sequencing workflow verification

• MSI algorithm evaluation

• Bioinformatics pipeline testing

• Cross-platform comparison

• Long-term quality monitoring

5. Multiple Sample Types for Broader Validation Coverage

A strong MSI reference standard set should provide more than one positive and one negative sample. Multiple samples allow laboratories to evaluate assay performance across different genetic backgrounds and tumor sources.

This is useful because MSI detection can be affected by sample characteristics, tumor origin, DNA quality and marker behavior.

A reference standard set with multiple MSI-H and MSS samples allows laboratories to design a more complete validation study and better assess workflow robustness.

CB-Gene MSI Reference Standards: 12 Paired Samples for MSI Validation

CB-Gene provides MSI Reference Standards designed for PCR-CE and NGS-based MSI assay validation.

The CB-Gene MSI Reference Standard set includes 12 pairs of genomic DNA reference materials. Each pair contains a tumor-derived gDNA sample and a corresponding normal or reference gDNA sample.

The product set includes:

This 12-pair design gives laboratories both MSI-H and MSS materials for assay validation, method comparison and quality control.

The MSI status of these reference standards is confirmed by PCR-CE assay and NGS assay, making them suitable for laboratories working with either fragment analysis or sequencing-based MSI detection.

How CB-Gene MSI Reference Standards Support PCR-CE Validation

For PCR-CE workflows, CB-Gene MSI Reference Standards can be used to evaluate whether the assay can detect expected microsatellite marker changes.

The standards are suitable for PCR-CE MSI analysis involving markers such as BAT-25, BAT-26, MONO-27, NR-21, NR-24 and NR-27.

Laboratories can use these materials to verify:

• PCR amplification performance

• Capillary electrophoresis fragment sizing

• Tumor-normal comparison

• Marker instability patterns

• MSI-H classification

• MSS classification

• Run-to-run consistency

Because the set includes both MSI-H and MSS samples, it can support a more balanced validation design than using a single control material.

How CB-Gene MSI Reference Standards Support NGS Validation

For NGS workflows, CB-Gene MSI Reference Standards can be used to evaluate both wet-lab and dry-lab performance.

In the wet-lab process, the materials can help assess DNA input, library preparation, sequencing quality and panel performance.

In the dry-lab process, the same materials can help evaluate whether the MSI calling algorithm correctly classifies known MSI-H and MSS samples.

These standards can support NGS validation activities such as:

• Targeted gene panel validation

• MSI calling algorithm verification

• Bioinformatics pipeline optimization

• Inter-run reproducibility studies

• Cross-platform comparison between PCR-CE and NGS

• Routine quality control for sequencing-based MSI detection

For laboratories developing NGS-based MSI detection, using reference standards confirmed by both PCR-CE and NGS provides an additional layer of confidence.

Suggested Validation Design Using MSI Reference Standards

A practical MSI validation workflow may include the following steps.

Step 1: Test MSS Reference Standards

Start by testing MSS materials to confirm that the assay does not generate false MSI-H calls.

This step helps evaluate assay specificity and baseline stability.

Step 2: Test MSI-H Reference Standards

Next, test MSI-H materials to confirm that the assay can detect microsatellite instability.

This step helps evaluate assay sensitivity and positive classification performance.

Step 3: Repeat Across Multiple Runs

Testing the same reference standards across multiple runs helps evaluate reproducibility.

This is useful for monitoring variation caused by different days, operators, reagent lots or instruments.

Step 4: Compare PCR-CE and NGS Results

For laboratories using both methods, the same reference materials can be used to compare PCR-CE and NGS classifications.

This can help support method correlation and platform transition studies.

Step 5: Use Reference Standards for Routine QC

After validation, MSI reference standards can continue to be used as positive and negative controls in routine quality control programs.

Conclusion

Selecting the right MSI reference standard is essential for reliable PCR-CE and NGS assay validation. Laboratories need well-characterized MSI-H and MSS materials to evaluate assay accuracy, reproducibility, classification performance and workflow stability.

CB-Gene MSI Reference Standards provide 12 paired genomic DNA reference materials, including MSS-P1 to MSS-P4 and MSI-H-U1 to MSI-H-U8. The standards are confirmed by PCR-CE and NGS assays and are designed to support MSI assay validation, quality control, platform comparison and bioinformatics pipeline verification.

For laboratories and diagnostic developers working on MSI testing, CB-Gene MSI Reference Standards offer practical control materials for building reliable and reproducible MSI detection workflows.

Contact CB-Gene to learn more about MSI Reference Standards for PCR-CE and NGS assay validation.