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Standard Methods of Detection


Mononucleotide repeat microsatellite sequences are particularly sensitive to transcription errors, making them ideal targets for measuring microsatellite instability (MSI) using PCR amplification with fluorescently labelled primers. To detect MSI, the amplified fragments from the tumor and matched normal samples are measured and compared. The amplified fragments are separated by size using capillary electrophoresis, and the fluorescent labelling allows them to be separated by the color of the fluorescent tag. Multiplexing the fluorescent labels allows many different fragments of similar size to be detected in the same reaction. Changes in sizing indicate that there is microsatellite instability, and tumors that contain this microsatellite instability are referred to MSI-high or MSI-H.

detecting msi by pcr


Immunohistochemistry (IHC) is a histological technique using labeled, protein-specific antibodies to detect proteins. Each protein is detected using a separate tissue sample. In the case of the major mismatch repair proteins, tumors that have loss of one or more of the mismatch repair proteins are referred to as dMMR, because deficiency in mismatch repair is implied.

Detecting mmr by ihc


Immunohistochemistry measures the presence or absence of MMR proteins; however, the presence of MMR protein expression is not necessarily a conclusive measure of MMR function. There can be a loss in the function of these proteins without a corresponding loss of the protein in the cell, with 5–10% of proteins retaining antigenicity when they are not functional (1).

MSI by PCR directly measures changes in DNA caused by loss of MMR protein function as opposed to measuring the proteins themselves as in IHC. This makes MSI a functional measure of mismatch repair deficiency that detects loss in MMR repair function, regardless of cause.

Alternative Methods for Detection

MSI analysis using next generation sequencing

MSI analysis by next generation sequencing (NGS) determines sequences of microsatellites and then compares them to either matched normal tissue or a consensus sequence. Differences in the microsatellite sequence length indicate instability. Algorithms are used to determine if the amount of instability detected is significant, and samples are assigned as MSI-H or MSS (microsatellite stable). In some cases, computational challenges inherent to NGS analysis of microsatellite homopolymers may mean no call can be made. There is currently no consensus on which loci or what data analysis pipeline and cutoffs should be used for MSI determination by NGS so there are many different approaches used in the field.

Comparison of MSI by PCR, IHC and NGS



Detects the functional failure of tumor MMR proteins, resulting in changes (instability) in microsatellite allele length. Detects the presence or absence of tumor MMR proteins using monoclonal antibodies against the major mismatch repair genes Detects mutations within microsatellite sequences of tumor samples


$44/ sample

$50–70/slide (x 4 slides)

$1K–3K/samples (variable)


Up to 96 samples (med to high)

Low to medium

Variable (more information = lower throughput)

Sample Input

1ng (<1 FFPE curl)

4-5 slides

10–20 slides


2–3 days

1–3 days

2–3 weeks

False Negative Rate

0.3–4% (2)

5–10% (3)

Variable (not standardized)



No (must do separate slides)


Markers Analyzed

5 mononucleotide loci (NCI recommended, Revised Bethesda Panel) (4)

4–5 MMR proteins (MSH6, MSH2, PMS2, MLH-1 & BRAFv600E (optional)

No standard panel or analysis method established


Functional assay, low false negative rate, time tested, NCI recommended standardized loci panel available

Shows which genes to investigate, familiar technology

Provides more information besides just MMR (MSI)


Only characterizes MSI, does not show which MMR gene to investigate

Consumes multiple tissue sections, 5–10% false negative rate, uses more sample (3)

No standardized panel, must sequence a large panel to be cost effective, technically challenging, expensive, difficult to troubleshoot, consumes more sample than IHC or PCR


  1. Funkhouser, W.K. Jr. et al. (2012) J. Mol. Diagnostics 14, 91–103.
  2. Based on an internal analysis of publications comparing MSI-PCR v. IHC-dMMR in colorectal cancer from 2004–2018. Literature bundle available from Promega Medical Affairs upon request.
  3. Sepulveda, A.R. et al. (2017) Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology. J. Mol. Diag. 19, 187–225.
  4. Umar et al. (2004) J. Natl. Cancer Inst. 96, 261–8.