Supplemental file to:
“Assessment of promoter methylation and expression of SIX2 as a diagnostic and prognostic biomarker in Wilms' tumor” by Song et al.
Methylation-specific PCR (MSP)
Genomic DNA was extracted from tissues using DNAiso Reagent and Blood Genome DNA
Extraction Kit (Takara, Tokyo, Japan) according to the manufacturer’s instructions. The quantity and quality of the extracted DNA was described by using NanoDrop 2000 spectrophotometer
(Thermo scientific, USA). The genomic DNA was treated with sodium bisulfate using EZ DNA
MethylampTM kit (ZYMO,USA),which converted the non-methylated cytosine residues to uracil,while methylated cytosine remained unchanged. Normal leukocyte DNA was methylated in vitro with SssI methylase(NEB,UK) to generate completely methylated DNA as a positive control.
Methylation of SIX2 gene promoter was determined by MSP.Primers distinguishing methylated (M) and unmethylated (U) alleles were designed by online software(http://www.urogene.org/methprimer/index1.html)to amplify the sequence, synthesized by (Sangon, Shanghai, China):
SIX2 M-forward: 5-ATGTTGTTTATTTTCGGTTTTACGT−3;
SIX2 M-reverse: 5-CGCTTTCATTCTTATAAAAATACTCG−3;
SIX2 U-forward: 5-ATGTTGTTTATTTTTGGTTTTATGT−3;
SIX2U-reverse: 5-ACTTTCATTCTTATAAAAATACTCACA−3.
Briefly, MSP amplification was carried out in a final reaction mixture of 25μL containing 100ng bisulfite-treated DNA, 5 pmoles each primer, 2.5μLdNTPs, 2.5μL10x PCR buffer, and 0.25μl
Blend Taq-Plus (Takara, Tokyo). PCR parameters were: 94°C x 2 min; (94°C x 30 sec,53°C × 30 sec, 72°C × 1 min) ×40 cycles; and 72°C for 5 minutes. During amplification positive and negative control samples were included. PCR products were resolved with 2% agarose gel electrophoresis, and band of methylated and/or unmethylated genes were visualized using the
GDS800 uv gel imaging(UVP,USA) .
Quantitative real-time PCR(qRT-PCR)
Total RNA was extracted from tissues using RNAiso Reagent (Takara, Tokyo, Japan) following kit’s instructions. The quality,quantity and integrity of extracted RNA was determined by
Nanodrop 2000 and agarose gel electrophoresis,in which all the isolated RNA samples had

260/280 ratio of 1.8-2.0.CDNA was prepared from 1µg RNA using PrimeScript RT reagent
Kit(Takara, Tokyo, Japan), quality of which was confirmed by β-actin gene.
QRT-PCR was performed to determine the amplification levels of SIX2. Primers used are included below: 1
SIX2-forward: 5-GCCGAGGCCAAGGAAAGGGAG-3;
SIX2-reverse: 5-GAGTGGTCTGGCGTCCCCGA-3;
β-actin-forward: 5-GATGAGATTGGCATGGCTTT-3;
β-actin-reverse: 5-CACCTTCACCGTTCCAGTTT-3.
QRT-PCR was performed in a total volume of 20μL including 2μL of cDNA, 0.8μL each primer,0.4μL ROX Reference Dye II and 10 μLof SYBR Premix Ex Taq II(Takara, Tokyo,
Japan).Reactions were run on 7500 Fast Real-Time PCR System(ABI,USA)using the universal thermal cycling parameters(95°C 30 sec, 40 cycles of 5 sec at 95°C and 34 sec at
60°C; melting curve: 95°C 15 sec, 60°C 1 min, 95°C 15 sec, 60°C 15 sec and continues melting).
Each sample was run in triplicate and β-actin was run in parallel to standardize the input DNA.
The amplification levels of SIX2 were expressed as relative expression using the comparative
∆∆Ct method. SDS 1.4 software (ABI) was used to calculate the ΔΔCt relative expression values, normalized to endogenous control β-actin gene and relative to amplification of selected venous blood of normal child.
1.
Murphy AJ, Pierce J, de Caestecker C, et al.
SIX2 and CITED1, markers of nephronic progenitor self-renewal, remain active in primitive elements of Wilms' tumor.J Pediatr Surq 2012; 47:1239-49.