The Leiden University Medical Centre (LUMC) cohort includes clinical, histopathological, and genetic information on 64 UM cases, enucleated between 1999 and 2008. Clinical information was collected from the Integral Cancer Center West patient records and updated in 2019. For each sample, part of the tumor was snap frozen with 2-methyl butane and used for mRNA and DNA isolation, while the remainder was embedded in paraffin after 48 hours of fixation in 4% neutral-buffered formalin and sent for histological analysis. Chromosome status was determined with the Affymetrix 250K_NSP-chip and Affymetrix Cytoscan HD chip (Affymetrix, Santa Clara, California, United States of America). RNA was isolated with the RNeasy mini kit (Qiagen, Venlo, The Netherlands) and mRNA expression was determined with the HT-12 v4 chip (Illumina, San Diego, California, United States of America). Statistical analyses of the LUMC cohort were carried out in SPSS, version 25 (IBM Corp). For survival analysis, Kaplan-Meier and log-rank test were performed with death due to metastases as endpoint. Cases that died of another or unknown cause were censored. The two subpopulations that were compared in each analysis were determined by splitting the total cohort along the median value of mRNA expression for each analyzed gene.
Ht 12 v4 chip
Manufactured by Illumina
Sourced in United States
The HT-12 v4 chip is a gene expression microarray platform designed by Illumina for comprehensive whole-genome expression profiling. It provides comprehensive coverage of well-characterized genes, gene candidates, and splice variants. The chip features 47,000 probe sets that target transcripts and allow for the simultaneous analysis of 12 samples on a single array.
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Lab products found in correlation
9 protocols using ht 12 v4 chip
1
Genetic and Clinical Analysis of Uveal Melanoma
2
RNA Microarray Data Preprocessing
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Pre-processing of RNA microarray data relied on the intensities of 47,323 transcripts derived from Illumina BeadStudio without background correction and normalization measured in 1,029 individuals of the Sorbs cohort. Steps for pre-processing comprised 1. filtering of individuals with atypical low number of expressed genes (median - 3 interquartile ranges (IQR) of the cohort's values), 2. quantile normalization and log2- transformation, 3. filtering individuals with atypical gene-expression profiles (Euclidian distance to average expression larger than median +3 IQR), 4. filtering individuals with atypical values of internal quality parameters (quantified as Mahalanobis distance of quality control probes included on the HT-12 v4 chip by Illumina, individuals having a larger value than median +3 IQR of this measure were excluded), 5. correcting for batch effects on the basis of hybridisation chip numbers using Empirical Bayes estimates [34] (link), 6. linear adjustment for age, sex, and lymphocyte and monocyte cell counts. A total of 924 individuals fulfilled all quality criteria. For 898 of these, SNP array data were also available for eQTL analyses.
3
Chromosome Aberrations in Uveal Melanoma
4
Genetic Analysis of Chromosome 3 and 8q
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The QIAmp DNA Mini Kit was used to isolate DNA for single nucleotide polymorphism (SNP) analysis according to guidelines of the manufacturer (Qiagen, Venlo, The Netherlands). Status of chromosome 3 was determined with SNP analysis performed with the Affymetrix 250K_NSP chip and the Affymetrix Cytoscan HD chip (Affymetrix, Santa Clara, CA, USA) [14 (link)]. The copy number of chromosome 8q was identified with ddPCR. A threshold of >2.1 was defined as gain of 8q [14 (link)]. The RNeasy Mini Kit was used to isolate mRNA for gene expression analysis (Qiagen, Venlo, The Netherlands). Gene expression levels were obtained using the Illumina HT-12 v4 chip (Illumina, San Diego, CA, USA). Angiogenesis-related factors were selected based on literature regarding angiogenesis. Only these predefined genes were assessed in the current analysis (Table S5 ).
5
mRNA Profiling and HLA Class I Expression
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mRNA status was obtained from fresh frozen tumor tissue. RNA for gene expression profiling was isolated with the RNeasy mini kit (Qiagen). HLA Class I expression was measured on an Illumina HT-12v4 chip (Illumina) following the manufacturer’s guidelines. The probes seen in Table 5 were used for this analysis. They had previously been compared with immunohistochemical data [24 (link),28 (link)].
6
Gefitinib-Resistant PC-9 Cell Analysis
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Total RNA prepared from parental and Gefitinib‐resistant PC‐9 cells was used to probe Illumina HT‐12v4 chip. Data analysis was carried out with background correction using Genespring software and was average normalized. Test samples were considered to be differentially expressed when they crossed the threshold of detection P‐value ≤ 0.05 and differential score P value ≤ 0.05 among all the samples. Significantly differentially expressed genes were annotated with functional assignments to help determine which gene categories were enriched with differentially expressed genes based on the Gene Ontology (GO) database: biological process, molecular function, and cellular component; 1,000 differentially expressed genes were identified at a 1% false discovery rate by SAM analysis (Tusher et al, 2001 ).
7
Measuring PRAME Expression in Melanoma
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Preferentially expressed antigen in melanoma (PRAME) expression was available for 64 cases. messenger RNA expression was measured from archived snap-frozen material on an Illumina HT-12v4 chip (Illumina) using probe ILMN_1700031. Preferentially expressed antigen in melanoma expression was classified as positive or negative according to a cutoff value set at the inflection point of the PRAME expression curve obtained using probe ILMN_1700031.19 (link)
8
Profiling Angiogenesis and Immune Markers in Uveal Melanoma
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Messenger RNA was isolated from frozen tumour material for gene expression analysis using the RNeasy Mini Kit (Qiagen, Venlo, the Netherlands). The Illumina HT-12 v4 chip was used to determine gene expression levels (Illumina, San Diego, CA, USA). Hypoxia- and angiogenesis-related factors were selected based on the literature regarding angiogenesis in UM (Table S4 ). Probes for CD3, CD8, and CD68 were previously validated [28 (link)].
9
Quantifying Immune Infiltrates in Tissues
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Immune infiltrate was determined using immunofluorescence with antibodies against CD68 for macrophages (pixels/mm 2 ). 32 The mRNA gene expression was determined using the Illumina HT-12 v4 chip (Illumina) after mRNA isolation with an RNeasy Mini Kit (Qiagen). 33
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