Ils 600

cDNA products were subject to the same primer sequences and PCR conditions as mentioned above, with the exception of forward primer sequence used for this reaction was labeled with 5’ 56-JOEN fluorophore. The RT-PCR products amplified using JOE fluorophore labeled primer were subjected to fragment analysis on 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA) with the internal lane standard 600 (ILS 600) (Promega Corporation, Madison, WI), used as a DNA ladder to assign correct sizes to DNA fragments. The percentages of the different transcripts of the total transcripts were calculated based on the peak heights of individual fragments.

For the detection of microsatellite instability, a fluorescent PCR-based assay (MSI Analysis System Version 1.2, Promega) was used according to the manufacturer's instructions. The MSI Analysis System included fluorescently labeled primers for co-amplification of seven markers including five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) and two pentanucleotide repeat markers (Penta C and Penta D). Additionally, an Internal Lane Standard 600 (ILS 600, Promega) was used. In brief, 20 ng of genomic DNA was used for the PCR under following conditions: initial denaturation for 11 min at 95 °C and 1 min at 96 °C, ramp 100% to 94 °C for 30 s, ramp 29% to 58 °C for 30 s, ramp 23% to 70 °C for 1 min, for 10 cycles, followed by ramp 100% to 90 °C for 30 s, ramp 29% to 58 °C for 30 s, ramp 23% to 70 °C for 1 min, for 20 cycles, and final elongation at 60 °C for 30 min. The PCR products were separated by capillary electrophoresis using a 3500 Genetic Analyzer. Data were analyzed with the GeneMapperR software (Applied Biosystems) by comparing sample marker patterns with the pattern of a positive amplification control (K562 High Molecular Weight DNA). A shift of two or more mononucleotide markers was considered as MSI-high, a shift of one mononucleotide marker was considered as MSI-low.