Eco software

Methylation-sensitive high resolution melting (MS-HRM) was conducted only on those samples that showed positive results in MSP, to validate the observations. Whole genomic DNA was treated with sodium bisulfite using the Epitect Bisulfite Kit (Qiagen, Hilden, Germany) to convert unmethylated cytosines to uracils, following the manufacturer’s protocol, as described in [23 (link)]. Real-time PCR followed by HRM was carried out in high-performance Eco Real-Time PCR system (Illumina, San Diego, CA, USA). Primer sequences for MLH1 were described earlier [23 (link),24 (link)], while for MLH3 primers (Additional file 1: Table S1b) were designed using Methyl Primer Express Software v1.0 (Applied Biosystems, Foster City, CA, USA). The reaction mixture (10 μl final volume) consisted of 10 ng of template DNA, 1× EpiTect HRM Master Mix (Qiagen) and 300 nmol/l of each primers. PCR was initiated by incubation at 95°C for 5 min, followed by 50 cycles at 95°C for 10 sec, 56°C for 20 sec, 72°C for 10 sec. For each assay, a standard dilution series of EpiTect Control DNAs (Qiagen) was run to assess the quantitative properties and sensitivity of the assay. Fluorescence data were converted into melting peaks by the Eco Software (Illumina, Ver. 3.0.16.0). The cut-off value for aberrant methylation was set to 25% or higher.

The 2SYBR Select Mixture was used for quantitative real-time polymerase chain reactions on an Eco Real-Time PCR system (Applied Biomiga). Each 50-μl reaction mixture contained 2 μl cDNA template, 25 μl of SYBR Mixture, 1 μl of each primer (10 μM), and 21 μl double-distilled water. The temperatures were as follows: 50°C for 2 min; 95°C for 10 min; and 40 cycles of 95°C for 15 s and 60°C for 1 min. Dissociation curves were generated for each reaction to ensure specific amplification. Cycle threshold (CT) values were generated from applied Illumina Eco Real-time PCR software (Illumina). The relative expression level was determined using the ^2-ΔΔCt method (Rajwade et al., 2010 (link)) with the GAPDH gene as a reference. To validate the specificity of the amplification, a melting curve was constructed for each primer pair. Data were analyzed using Eco software (Illumina). Two technical replicates of each were performed.

After quantification of total RNA isolated from each tissue by using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Yokohama, Japan), reverse transcription was performed using the PrimeScript RT reagent Kit (Perfect Real Time) (Takara Bio) as the manufacturer’s instructions. Real-time quantitative PCR were then run on the Eco Real Time PCR System (Illumina, San Diego, CA) using Fast SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA), with the following gene-specific primers: HRBP-Fw and HRBP-Rv for HRBP and 18SrRNA-S and 18SrRNA-A for 18S rRNA, which was analyzed as the housekeeping gene. Data were analyzed using Eco Software (ver. 3.0, Illumina). A standard curve was generated using a serial dilution of plasmid harboring each gene.