Microsatellites are genetic motifs consisting of short nucleotide sequences repeated up to 100 times and comprising approximately 3% of the human genome. During replication, these regions are particularly susceptible to mutations, normally corrected by the DNA mismatch repair (MMR) system. Microsatellite instability (MSI) refers to the accumulation of uncorrected microsatellite errors which are used for indirect evaluation of MMR deficiency. Microsatellite instability-high (MSI-H) cancers are characterized by the presence of mutations in 30% or more of the microsatellites tested with implications for diagnosis and treatment planning.
MicroSight® MSI - easy and fast evaluation of microsatellite instability by real-time PCR
MicroSight® MSI is a new, simple and fast way of evaluating microsatellite instability (MSI) status of human DNA. The analysis is based on PentaBase’s proprietary INA® technology that enables fast and easy detection of microsatellite instability status by real-time PCR and high-resolution melt (HRM) analysis. MicroSight® MSI requires very little hands-on time and enables evaluation of patient microsatellite status of purified human genomic DNA in less than one and a half hours for fast treatment guidance of cancer patients.
MicroSight® MSI workflow. Only three fast and simple steps are needed for the analysis of microsatellite instability with MicroSight® MSI. The method is based on high-resolution melt analysis and gives a clear and objective evaluation of microsatellite status.
Evaluation of well-established MSI markers in paired and unpaired tumour samples
MicroSight® MSI PentaBase Panel evaluates instability of the well-established MSI markers BAT25, BAT26, NR22, NR24 and MONO27 allowing for easy conversion from current methods based on capillary electrophoresis. MicroSight® MSI can be used for analysis of both paired and unpaired tumour samples. Evaluation of unpaired tumour samples is done by comparison to a universal reference assay supplied by PentaBase (Strip HR). Up to three unpaired tumour samples can be analysed in each real-time PCR run.
Evaluation of microsatellite instability by MicroSight® MSI requires special software for analysis of HRM data which is supported by PentaBase's BasecCycler™ as well as the MyGo Pro (IT-IS Life Science Ltd.) real-time PCR instruments.
PlentiPlex™ MSI - multiplexed PCR assays for MSI fragment length evaluation by capillary electrophoresis
PlentiPlex™ MSI assays offer two different multiplexed MSI assays for the length analysis of five different microsatellite loci and evaluation of MMR status on DNA sequencer instruments:
- The mono- and dinucleotide panel recommended by the Bethesda guidelines (Bethesda panel)
- A mononucleotide panel detecting BAT-25, BAT-26, MONO-27, NR-22 and NR-24 loci (PentaBase panel)
Evaluation of MSI status using PlentiPlex™ MSI is done by comparing capillary gel migration charts (electropherograms) of the tumour samples with those of the germ-line (reference) DNA samples. Below is shown two examples of microsatelitte instability samples. The tumor tissues (bottom) are compared to non-tumor tissues (top) and MSI peaks are marked with red brackets.