MSI Testing (Colorectal Cancer)

MSI Analysis System (Research Use Only kit, Version 1.2, Promega Corporation). 

The MSI Analysis System is a multiplex, fluorescent PCR-based assay for detection of microsatellite instability. The MSI Analysis System includes fluorescently labeled primers for co-amplification of five quasimonomorphic mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). The PCR products are separated by capillary electrophoresis (CE) using an Applied Biosystems 3500 Dx Genetic Analyzer. Microsatellite instability at 2 or more mononucleotide loci is interpreted as MSI-high; microsatellite instability at a single mononucleotide locus or no instability at any of the loci tested is interpreted as microsatellite stable (MSS). Typically, MSI-high samples show instability at all loci with an average decrease in product size of 5 to 9 bases. The presence of MSI is detected with an analytical specificity of at least 99% and a detection limit of at least 5%.

Clinical Implication

Microsatellite instability (MSI) results from the systematic accumulation of deletions/insertions in short repetitive DNA sequences in tumor cells with a deficient mismatch repair (MMR) system. MSI occurs in approximately 15% of all colorectal cancers and is clinically useful to identify patients with hereditary nonpolyposis colorectal cancer (HNPCC, Lynch Syndrome) caused by germline mutations of MMR genes. MSI status may also predict cancer response/resistance to certain chemotherapies. 

In an attempt to standardize MSI analysis, a 1997 National Cancer Institute (NCI) workshop recommended a reference panel of five microsatellite markers for the detection of MSI and established MSI classification guidelines based on the results. The reference panel, referred to as the Bethesda panel, consists of two mononucleotide loci (Big Adenine Tract or BAT-25 and BAT-26) and three dinucleotide loci (D2S123, D5S346 and D17S250). Using the Bethesda panel, cancers with instability at 2 or more of these loci were interpreted as MSI-high, and cancers with no instability at any of the five loci were considered Microsatellite Stable (MSS). At a further follow-up NCI workshop, it was recognized that the sensitivity and specificity of the original Bethesda panel might be limited due to the inclusion of dinucleotide repeats, which are less sensitive and specific than mononucleotide repeats for the identification of cancers with MMR deficiencies. It has been suggested that a panel of five quasimonomorphic mononucleotide repeats, as used in the MSI Analysis System (Promega Corporation), may be sensitive and specific enough for the detection of MSI-high cancers and may obviate the need for normal matching DNA for the tumors being tested (1-8). An important design feature of the MSI analysis System is the spacing between the five different loci, which generally exceeds the typical number of bases shifted (average 5 to 9 bases) when MSI is present. The longest PCR amplicon size is 154 base pairs, which is advantageous for the analysis of formalin-fixed, paraffin-embedded tissue (FFPE). 

Specimen requirements

Acceptable specimens for the assay are formalin-fixed, paraffin-embedded colorectal carcinoma tissue specimens with a fixation time of 6-48 hours.


1 representative paraffin block is preferred. Alternatively, for resection specimens a minimum of 5 unstained tissue sections (5 µm thick) is required.

Storage and shipment instructions

Maintain and ship specimens at ambient temperature.


Insufficient tumor content may not allow the detection of MSI instability: 10% of tumor cells is required. Tumor content is evaluated prior to analysis and macrodissection is performed. Fixatives other than formalin or prolonged fixation time may give rise to inadequate results.

Special requirements



Five to 7 business days for respectively slides and paraffin blocks. 


  1. Umar A, Boland CR, Terdiman JP et al. Revised Bethesda Guidelines for Hereditary Nonpolyposis Colorectal cancer (Lynch Syndrome) and Microsatellite Instability. Journal of the National Cancer Institute 2004;96(4):261-268.
  2. De la Chapelle A, Hampel H. Clinical Relevance of Microsatellite Instability in Colorectal Cancer. Journal of Clinical Oncology 2010;28(20):3380-3387.
  3. Bacher JW, Flanagan LA, Smalley RL et al. Development of a fluorescent multiplex assay for detection of MSI-High tumors. Disease Markers 2004; 20:237-250.
  4. Murphy KM, Zhang S, Geiger T et al. Comparison of the Microsatellite Instability Analysis System and the Bethesda Panel for the Determination of Microsatellite Instability in Colorectal Cancers. Journal of Molecular Diagnostics 2006;8(3):305-311.
  5. Anderson S, Bloom KJ, Vallera DU et al. Multisite Analytic Performance Studies of a Real-Time Polymerase Chain Reaction Assay for the Detection of BRAF V600E Mutations in Formalin-Fixed Paraffin-Embedded Tissue Specimens of Malignant Melanoma. Arch pathol lab Med 2012;136(11):1385-1391.
  6. Buhard O, Suraweera N, Lectard A et al. Quasimonomorphic mononucleotide repeats for high-level microsatellite instability analysis. Disease Markers 2004;20:251-257.
  7. Patil DT, Bronner MP, Portier BP et al. A five-marker panel in a multiplex PCR accurately detects microsatellite instability-high colorectal tumors without control DNA. Diagn Mol Pathol 2012 Sep;21(3):127-33.
  8. Berg KD, Glaser CL, Thompson RE et al. Detection of microsatellite instability by fluorescence mulitplex polymerase chain reaction. J Mol Diagn 2000, 2:20-28.