Humanht 12 v3 expression beadchip

The gene expression profiles of H292 and H292-Gef cells were determined using Illumina HumanHT-12 v3 Expression BeadChip (Illumina, Inc., San Diego, CA, USA) according to the technical manual of Macrogen (Seoul, Korea). Total RNA was extracted from cells with TRI reagent (Invitrogen, Grand Island, NY, USA) following the manufacturer’s instructions. The intensity and quantity of total RNA was assessed using a Nanodrop ND-1000 spectrometer (Nanodrop Technologies, Wilmington, DE, USA), and 0.55 μg of total RNA was used to prepare biotinylated cRNA using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX, USA). After fragmentation, 0.75 μg of labeled cRNA were hybridized to the Illumina HumanHT-12 Expression BeadChip following the manufacturer’s protocols. Arrays were scanned with the Illumina Bead Array Reader Confocal Scanner. Array data export processing and analysis was performed using Illumina GenomeStudio v2009.2 (Gene Expression Module v1.5.4).

Expression levels for ARF and INK4a were quantified from total RNA (100 ng) of 29 LXSCC tumors using real‐time reverse transcriptase‐PCR (qRT‐PCR) Taqman^® probes and the TaqMan RNA‐to‐Ct^™ 1‐Step Kit (Applied Biosystems, Waltham, MA). Resulting cycle threshold (Ct) values were normalized to GAPDH and subsequently converted to relative expression (2^−ΔΔCT) values.
As an additional platform, whole‐genome expression microarray data from a prior analysis of head and neck tumors were also assessed 11 and 21 of the LXSCC samples used for the methylation analysis were used in this analysis. TRIzol (Ambion, Austin, TX) extracted RNA (500 ng) for each tumor sample was amplified and biotin labeled using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX). Messenger RNA expression levels were subsequently analyzed by RNA hybridization to the Illumina HumanHT‐12‐v3 Expression BeadChip (Illumina, San Diego, CA). Microarray probes for the CDKN2A gene were identified using the RefSeq database release 17 and UniGene build 188. RNA expression levels were assessed by a probe targeting ARF and a combined CDKN2A probe. In addition, a probe corresponding to the INK4b gene in the adjacent CDKN2B region was also identified and used to measure corresponding expression of that transcript.

In the Rotterdam Study, RNA was isolated from whole blood and gene expression profiling was performed using the IlluminaHumanHT-12v4 Expression Beadchips (Illumina, San Diego, CA, USA). The expression dataset is available at Gene Expression Omnibus (GEO) public repository under the accession GSE33828: 881 samples are available for analysis. In KORA F4, total RNA was extracted from whole blood and the Illumina Human HT-12 v3 Expression BeadChip (Illumina, San Diego, CA, USA) was used for gene expression profiling [59 (link)]. A more detailed description is implemented in Additional file 8.
In the current study, genes of interest were selected using a previous TWAS testing the association between gene expression and current versus never-smoking status [8 (link)]. In this TWAS, the meta-analysis was performed on all transcripts with matching gene Entrez IDs. Employing a significance threshold of FDR < 0.05, 886 significant gene Entrez IDs were identified, of which 387 replicated in an independent dataset. Employing the annotation file provided by the Illumina (HumanHT-12_V4), we found 502 gene expression probes to be annotated to these gene Entrez IDs out of which 443 were present in the Rotterdam Study and were included in the current study (Additional file 8: Table S9).