Microsatellite Instability Analysis Protocol

EMAST status was determined using 5 polymorphic tetranucleotide markers (UT5037, D9S242, D20S85, D8S321, and D20S82) [22 (link)]. Genomic DNA extracted from tumors and counterpart normal tissues were PCR-amplified by specific primers for each tetranucleotide marker using Platinum PCR Supermix (Invitrogen, USA) as per the manufacturer’s protocol. PCR was performed using 6-FAM or HEX labeled primers. Cycling conditions were as follows: 95°C for 15 minutes for the initial heat activation; 40 cycles of 94°C for 1 minute, 55 C°–62C° for 1 minute, and 72°C for 30 seconds; and a final extension at 72°C for 10 minutes. Fluorescently labeled fragments generated by PCR were analyzed on an Applied Biosystems 3730xl DNA Analyzer with the GeneMarker (SoftGenetic LLC, PA). PCR products were used for DNA fragment analysis to identify frameshift mutation at tetranucleotide repeats for each locus. When aberrant peaks +/- multiples of 4 nucleotides were observed in the electrophoretograms from tumor as compared to that of the paired non-tumor, the marker was listed positive for frameshift instability. Tumors showing frameshifts in at least 2 tetranucleotide markers compared to paired normal tissue were categorized as EMAST-positive tumors, whereas all others were categorized as EMAST-negative tumors.