Abi prism 7900 ht platform

Genomic DNA was extracted from whole blood samples collected in an EDTA tube. Single nucleotide polymorphisms (SNPs) in the IRS1 (rs1801278, G > A) [GeneBank: NM_005544], and PPARG (rs1801282, C > G) [GeneBank: NM_015869] were genotyped using high throughput real-time polymerase chain reaction (RT-PCR) in two independent laboratories (Centre for Proteomic and Genomic Research Institute of infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town and Obesity and Chronic Diseases of Lifestyle, Faculty of Health & Wellness Sciences, Cape Peninsula University of Technology) on the ABI Prism 7900HT platform(Applied Biosystems, USA) and a BioRad Optica (BioRad, USA) using Taqman genotyping assay (Applied Biosystems, USA). Direct sequencing was used to for analytical validation of high throughput genotyping against direct sequencing as the gold standard

Total RNA was prepared using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA by RevertAid cDNA Synthesis Kit (RR047A, Takara, Tokyo, Japan). PCR amplification was conducted on an ABI Prism 7900HT platform (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Mix (04913850001, Roche, Basel, Switzerland). β-actin was chosen as endogenous control. Relative quantification was assessed by the 2^-∆∆Ct method. The sequences of primers used are listed in Table 1.Table 1

The sequences of primers for qRT-PCR

Gene	Primer sequence
FOXO3a
Forward	5′-CGGACAAACGGCTCACTCT-3′
Reverse	5′-GGACCCGCATGAATCGACTAT-3′
SPRY2
Forward	5′-CTCGGCCCAGAACGTGATT-3′
Reverse	5′-GGCAAAAAGAGGGACATGACAC-3′
β-catenin
Forward	5′-CATCTACACAGTTTGATGCTGCT-3′
Reverse	5′-GCAGTTTTGTCAGTTCAGGGA-3′
TCF4
Forward	5′-AGAAACGAATCAAAACAGCTCCT-3′
Reverse	5′-CGGGATTTGTCTCGGAAACTT-3′
E-cad
Forward	5′-CGAGAGCTACACGTTCACGG-3′
Reverse	5′-GGGTGTCGAGGGAAAAATAGG-3′
VIM
Forward	5′-GACGCCATCAACACCGAGTT-3′
Reverse	5′-CTTTGTCGTTGGTTAGCTGGT-3′
β-actin
Forward	5′-ATCACCATTGGCAATGAGCG-3′
Reverse	5′-TTGAAGGTAGTTTCGTGGAT-3′

A total of 734 SNPs located in the coding regions and untranslated-regions (5′-UTR and 3′-UTR) of six CELF members were retrieved from the International HapMap database (Rel28, PhaseII+III, August 10, Build 36; http://hapmap.ncbi.nlm.nih.gov/) and the NCBI database (dbSNP version 126, http://www.ncbi.nlm.nih.gov), if their minor allele frequencies (MAF) in Chinese Han population (CHB) were above 5%. Subsequently, 114 tagging SNPs were identified by linkage disequilibrium (LD) analysis using Haploview software (version 4.2; r² < 0.5).
Venous blood sample was collected from each patient prior to any treatment. DNA was extracted from blood samples using the QIAamp DNA Blood Midi Kit (QIAGEN, Valencia, CA). In discovery stage, candidate SNPs were genotyped in 717 NPC patients by using GoldenGate^® Genotyping Assay (Illumina Inc, San Diego, CA) according to manufacturer's instructions. Two SNPs were excluded due to genotyping failure (call rates < 95%). The overall call rates ranged from 98.7% to 100% for the remaining 112 SNPs. Five promising SNPs with significant P-value (<0.05) in discovery stage were further genotyped in the validation sample of 1, 520 NPC patients, using TaqMan assay on ABI PRISM 7900 HT platform (Applied Biosystems Inc.). The details of primers and probes were shown in Supplementary Table S3.

Total RNA was reverse-transcribed by RevertAid cDNA Synthesis Kit (K1622, Thermo Fisher Scientific, Waltham, MA, USA) and amplified on an ABI Prism 7900HT platform (Applied Biosystems, Foster City, CA, USA) with specific miRNA primers (Table 2). U6 snRNA or GAPDH was used as endogenous control. The comparative Ct method was employed to estimate the relative expression levels.

Genomic DNA was extracted from whole blood samples collected in an EDTA tube. Single nucleotide polymorphisms (SNPs) in the PPARG, Pro115Gln (rs1800571, G>T), Val290Met (rs72551362, G>A), Pheu388Leu (rs72551363, T>A), Arg397Cys (rs72551364, C>T), and His447His (rs3856806, C>T), were genotyped using high throughput real-time polymerase chain reaction (RT-PCR) in two independent laboratories on the ABI Prism 7900HT platform (Applied Biosystems, USA) and a Bio-Rad Optica (BioRad, USA) using TaqMan genotyping assay (Applied Biosystems, USA). Conventional polymerase chain reaction followed by direct DNA sequencing was performed for analytical validation of high throughput genotyping.

Quantitative RT-PCR was performed using the SYBR® Premix Ex TaqTM Kit (TaKaRa, Japan) on an ABI PRISM 7900HT platform (Applied Biosystems, USA), according to the manufacturer’s instructions. For each sample, two biological replicates were analyzed by three technical repeats. The rice ubiquitin-1 gene (LOC_Os03g13170) was used as the internal reference gene. The primers for qRT-PCR analysis are listed in Table S5.