Polymerase chain reaction (PCR) conditions to measure GAA•TTC repeat length were described previously.21 (link) Quantitative reverse transcriptase PCR (qRT-PCR) was performed using the qScript One-Step SYBR Green qRT-PCR kit from Quanta Biosciences (Gaithersburg, MD) according to the manufacturer’s instructions. qRT-PCR reactions were detected on a PTC-200 thermal cycler with the Chromo4 real-time module (MJ Research, Waltham, MA). Primers to detect FXN mRNA were previously described.6 (link) Primers for neuronal characterization were as follows: MAP2-R1 (5′-CAGGAGTGATGGCAGTAGAC-3′), MAP2-F2 (5′-TTTGGAGAGCATGGGTCAC-3′) for the MAP2 gene, HUC-F1 (5′-GGTTCGGGACAAGATCACAG-3′), and HUC-R1 (5′-CTGAACTGGGTCTGGCATAG-3′) for the ELAVL3 gene. qRT-PCR primers for pluripotency mRNA markers were as previously published.20 (link) Neuronal cell gene expression profiling was performed on Illumina (San Diego, CA) HT12 arrays, and statistical analysis was as described in Ku et al.20 (link)
Ht12 arrays
Manufactured by Illumina
The HT12 arrays are a high-throughput gene expression profiling platform developed by Illumina. These arrays are designed to measure the expression levels of thousands of genes simultaneously across multiple samples. The HT12 arrays provide a comprehensive and efficient way to analyze gene expression patterns and identify differentially expressed genes in a wide range of biological studies.
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5 protocols using ht12 arrays
1
Quantitative PCR for Friedreich's Ataxia
2
Melanoma Cell Gene Expression Analysis
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The gene expression array dataset [22 (link)] and pre-processing methods [48 (link)] have been previously described. Briefly, RNA was purified from melanoma cell lines (Qiagen AllPrep kits) and assayed using Illumina HT12 arrays following the manufacturer’s protocols. Hierarchical clustering was performed in Partek Genomic Suite using Euclidian distance and average linkage.
3
RNA Quality Assessment and Microarray Analysis
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The RNA quality and yield were assessed with an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and a NanoDrop Technologies ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All microarray experiments were performed in one batch. Two hundred nanograms of total RNA were amplified and purified using a TotalPrep RNA Amplification Kit (Applied Biosystems/Ambion, Foster City, CA, USA). The amplified complementary DNA was hybridized on Illumina HT-12 arrays, and the data were extracted with Illumina Genome studio software. Pathway analysis was performed using BRB-ArrayTools (National Cancer Institute, USA). Over-represented Biocarta pathways were identified using Efron-Tibshirani’s GSA test p <0.005. Efron-Tibshirani’s test uses ‘maxmean’ statistics to identify gene sets differentially expressed. All heatmaps show unsupervised hierarchical analysis results (data have been submitted to Gene Expression Omnibus (GEO) public repository, accession number GSE73355).
4
Differential Gene Expression in Breast Cancer
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Gene expression of 1904 samples from METABRIC (299 TNBC and 1605 non-TNBC) using Illumina HT-12 arrays were downloaded [19 (link)]. Whole-transcriptome sequences of 1091 primary breast cancer (115 TNBC and 976 non-TNBC) and 112 normal cases in TCGA were downloaded in the format of ‘illuminahiseq_rnaseqv2-RSEM_genes_normalized (MD5)’ from FireBrowse [18 (link)]. Boxplots for gene expression comparisons in METABRIC [19 (link)] and TCGA [18 (link)] datasets were created using the R “ggplot” package. TCGA data were transformed to log2 of the gene expression value plus 1, to make them the same scale as the METABRIC gene expression data. P-values were independently calculated using Wilcoxon and ANOVA tests to compare between two, and among multiple groups, respectively.
5
Differential Gene Expression Analysis in Breast Cancer
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