7500 real time polymerase chain reaction system

From human samples, 30 mg of tissue was homogenized in RLT buffer containing β-mercaptoethanol. Complementary DNA (cDNA) was manufactured using the SENSIFast kit (Bioline) using 1 μg mRNA, as per the manufacturer’s instructions. From the cell lines, mRNA was purified using RNeasy kits (Qiagen, Manchester, UK) as per the manufacturer’s instructions. mRNA samples were reverse transcribed to form cDNA using the Tetro cDNA Synthesis Kit (Bioline Reagents Ltd., London, UK).
Expression of specific mRNAs was determined on a 7500 real-time polymerase chain reaction system (Applied Biosystems) using the QuantiTect Probe reverse transcription polymerase chain reaction kit (Qiagen). Relative expression was determined using the 2^−∆∆Ct method. The Taqman assays are described in Supplemental Table 2.

Because the metabolism of acetaldehyde, a well‐established carcinogen generated through alcohol metabolism, is determined by the genotype of the ALDH2 gene, we decided to investigate whether the health inequality in the association between alcohol and HNC risk is more prominent among genetically susceptible individuals. ALDH2 has a well‐known functional single nucleotide polymorphism (SNP), rs671.¹³ The ALDH2*1/*2 genotype and the ALDH2*2/*2 genotype encode an enzyme with less than 50% and 4% enzyme activity, respectively, compared to the enzyme encoded by the ALDH2*1/*1 WT genotype.¹⁴ALDH2 rs671 was genotyped for each study participant using the TaqMan‐based allelic discrimination method on a 7500 Real‐Time Polymerase Chain Reaction System (Applied Biosystems). To minimize genotyping error, 10% of the samples were randomly chosen for duplicate genotyping and the results showed 100% concordance.

Genotyping of two TLR2 SNPs, rs3804099 and rs3804100, and one TLR4 SNP, rs11536889, was accomplished with Taqman–based allelic discrimination method on an Applied Biosystems 7500 Real–Time Polymerase Chain Reaction System (Applied Biosystems, Foster City, CA). To detect genotyping errors, 10% of the samples were randomly selected for duplicate genotyping and a concordance of 100% was observed.

cDNA libraries were prepared using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1) (New England Biolabs, MA, USA) according to the manufacturer’s instructions with modifications on library size selection. Amplified cDNA libraries were electrophoresed on 12.5% polyacrylamide gel (E-T 12.5L, ATTO, Tokyo, Japan), and ~140 nucleotide bands (the length of adapter-ligated miRNA constructs) were eluted and precipitated by ethanol. The library sizes were checked by a bioanalyzer using the Agilent High Sensitivity DNA Kit (Agilent Technologies). The libraries were quantified by using a 7500 real-time polymerase chain reaction system (Applied Biosystems, CA, USA) with KAPA Library Quantification Kits (Kapa Biosystems, MA, USA) according to the manufacturer’s instructions. Furthermore, the libraries were equimolarly pooled for sequencing. Thereafter, sequencing was performed using MiSeq (Illumina, CA, USA) with single-end 36 cycles and 10% PhiX control (Illumina). The raw sequencing data are available from the database of the National Center for Biotechnology Information (SRA accession: SRP136572).

Total RNA was obtained from fresh tissue samples and cells by TRIzol reagent (Invitrogen, Life Technologies, Paisley, UK). The obtained RNA was reversed transcribed into single-stranded cDNA with PrimeScript reverse transcriptase kit (TaKaRa, Shiga, Japan) following the manufacturer’s guidelines. The expression level of miR-492 was determined by qRT-PCR with the SYBR green I Master Mix kit (Invitrogen, Carlsbad, California, USA) on a 7500 real-time polymerase chain reaction system (Applied Biosystems, USA). U6 was used as an endogenous control for miR-492. The sequences were as follows: miR-492 (F): 5′-GCCGAGAGGACCTGCGGGA-3′, miR-492 (R): 5′-CTCAACTGGTGTCGTGGA-3′; U6 (F): 5′-CTCGCTTCGGCAGCACA-3′, U6 (R): 5′-AACGCTTCACGAATTTGCGT-3′. The final expression value was calculated using the 2^−ΔΔCt method.

Total RNA was extracted by using a MicroElute Total RNA Kit (Omega Bio-Tek, Norcross, GA, USA). The comparative delta-delta threshold cycle (Ct) method was adopted to analyze gene products by using the SYBR Select Master Mix (Applied Biosystems) in a 7500 Real-Time Polymerase Chain Reaction System (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control gene to calculate the relative gene expression. The experiment was repeated three times, and the results were expressed as the mean ± standard deviation. The primer sequences are reported in Additional file 1: Table S3.