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Cat. No.: 2B3DMSI-50 (for 50T)

Cat. No.: 2B3DMSI-200 (for 200T)

Description

2B3D Microsatellite Instability Assay Kit by Fluorescent PCR and Capillary Electrophoresis, also known as the MSI Assay Kit or the 2B3D Microsatellite Instability Analysis Kit (MSI Analysis System), is designed for the rapid and highly sensitive detection of microsatellite instability in human cells using fluorescent PCR and capillary electrophoresis.

Microsatellites are short tandem repeat (STR) DNA sequences, 1-6 base pairs in length, widely present in the human genome, with repeat units of 10-50 times, including mono-, di-, or multi-nucleotide repeats. Microsatellite instability (MSI) refers to changes in the length of these DNA sequences caused by the insertion or deletion of repeat units, reflecting the frequency of DNA replication errors. Depending on the extent of MSI, it can be categorized into high-frequency microsatellite instability (MSI-H) and low-frequency microsatellite instability (MSI-L). MSI is primarily repaired by four mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, PMS2). Genetic and epigenetic (e.g., methylation) alterations in MMR genes can lead to loss of MMR protein function, resulting in MMR deficiency (dMMR) and causing MSI. MSI is closely related to tumorigenesis but varies widely among different tumors, with high incidence rates in endometrial cancer, colorectal cancer, and gastric cancer. MSI testing has significant clinical relevance for screening Lynch syndrome (an autosomal dominant disorder highly associated with colorectal and endometrial cancers), aiding in tumor diagnosis and classification, serving as an important prognostic and therapeutic marker for colorectal cancer, and predicting the efficacy of tumor immunotherapy. Additionally, MSI can be used for the clinical diagnosis of reproductive system defects caused by dMMR.

Methods for detecting MSI include immunohistochemistry (IHC) for MMR protein expression in tissue sections, and PCR to compare the sizes of specific microsatellite sequence amplification products between normal and tumor samples. In some cases, MMR proteins may lose their repair capability yet still bind to antibodies, potentially leading to false positives. In contrast, PCR results are more reliable with higher accuracy, sensitivity, and specificity.

Typically, MSI analysis requires the detection of at least five microsatellite loci. Authoritative guidelines recommend two panels: the 2B3D Panel, including two mononucleotide sites (BAT25 and BAT26) and three dinucleotide sites (D2S123, D5S346, and D17S250); and the 5D Panel, including five mononucleotide sites (BAT25, BAT26, NR21, NR24, and NR27). Both panels have been validated by extensive research data and provide reference value. We offer MSI assay kits based on both the 5D Panel and 2B3D Panel, allowing users to select the appropriate kit according to their research needs.

This kit, based on the 5D Panel, provides a mixture of five pairs of fluorescently labeled primers for multiplex amplification of the above five mononucleotide loci. Its specificity has been experimentally validated, as detailed in the table below.

Note: The allele fragment sizes listed in the table were obtained using the primers and reagents provided with this kit for multiplex amplification, followed by analysis with a 3730xl DNA Analyzer using a 50 cm capillary. When different amplification reagents or instruments are used, the allele fragment sizes may vary slightly.

The kit uses PCR and provides the necessary reagents for amplifying the microsatellite loci. After amplification, users can analyze the amplified products of the test and corresponding normal samples via capillary electrophoresis to generate allele profiles for comparison. Microsatellite instability is indicated by peak shifts or changes in the number of peaks (new alleles) in the test sample compared to the normal sample. Generally, if 40% or more of the microsatellite loci exhibit instability, it is classified as high-frequency microsatellite instability (MSI-H). If less than 40% of the loci show instability, it is classified as low-frequency microsatellite instability (MSI-L). For this kit, instability in 2 or more loci is considered MSI-H.

The kit also includes a PCR Control Template containing the marker genes for the five microsatellite loci mentioned above. This helps determine whether the kit is functioning correctly.

This kit only includes the reagents required for multiplex fluorescent PCR experiments. For capillary electrophoresis and analysis of the products, please contact tech@sbsbio.com.

Components

• MSI PCR Mix (2X)
• 2B3D MSI Primer Pair Mix (10X)
• Control Template
• Ultrapure Water