Table I.

Details of qPCR assays.

Assay	Primer sequences	Amplicon size/CpGs product (bp)	qPCR efficiency (%)	qPCR reaction conditions	qPCR reaction content
RASSF1A promoter methylation	5′-TTTTTTTAGGATTTAGATTGGG-3′ 5′-CCTACACCCAAATTTCCATTAC-3′	71/4	101	95°C, 5 min; 42× (95°C, 5 sec; 55°C, 10 sec; 72°C, 10 sec; 75°C, 10 sec, sample reading) Melting curve: 95°C, 15 sec; 60°C, 1 min; 60°C → 95°C reading every 0.1°C	5 μl SensiFast HRM (BioLine, London, UK), 300 nM each primer, ∑ 10 μl
RASSF1A mRNA level measurment	5′-CTCGTCTGCCTGGACTGTTGC-3′ 5′-TCAGGTGTCTCCCACTCCACAG-3′	101	98.5	95°C, 3 min; 37× (95°C, 5 sec; 58°C, 10 sec; 72°C, 10 sec; 75°C, 10 sec, sample reading) Melting curve: 95°C, 15 sec; 60°C, 1 min; 60°C → 95°C reading every 0.3°C	5 μl SensiFast NoRox SYBR-Green (BioLine), 200 nM each primer, ∑ 10 μl
RASSF1C mRNA level measurment	5′-TACTGCAGCCAAGAGGACTCGG-3′ 5′-TCAGGTGTCTCCCACTCCACAG-3′	116	92.3	95°C, 3 min; 37× (95°C, 5 sec; 58°C, 10 sec; 72°C, 10 sec; 75°C, 10 sec, sample reading) Melting curve: 95°C, 15 sec; 60°C, 1 min; 60°C → 95°C reading every 0.3°C	5 μl SensiFast NoRox SYBR-Green (BioLine), 200 nM each primer, ∑ 10 μl
GUSB mRNA level measurment for qPCR normalization	5′-ATGCAGGTGATGGAAGAAGTGGTG-3′ 5′-AGAGTTGCTCACAAAGGTCACAGG-3′	177	99.6	95°C, 3 min; 35× (95°C, 5 sec; 57°C, 10 sec; 72°C, 10 sec; 75°C, 10 sec, sample reading) Melting curve: 95°C, 15 sec; 60°C, 1 min; 60°C → 95°C reading every 0.3°C Melting curve: 95°C, 15 sec; 60°C, 1 min; 60°C → 95°C reading every 0.3°C	5 μl SensiFast NoRox SYBR-Green (BioLine), 200 nM each primer, ∑ 10 μl