The NCBI-Gene Expression Omnibus (GEO) database, a global public gene data repository run by the National Center for Biotechnology Information (NCBI) of the National Library of Medicine, provided the source data for this study (https://www.ncbi.nlm.nih.gov/geo/ ). The GEO data information includes genes, proteomic analyses, non-coding RNA analysis, etc. The majority of worldwide research organizations maintain these data with their original study findings. For this study, the GEO database’s two keloid-related datasets, GSE92566 and GSE7890 mRNA expression profiles (Homo sapiens), were retrieved. The authors of GSE92566 are Fuentes-Duculan J et al. It was created using the [HG-U133 Plus 2] Affymetrix Human Genome U133Plus2.0 array on the GPL570 platform. For this experiment, six samples from GSE92566 were selected, including two groups (three keloid lesions and three adjacent non-lesioned samples; in addition, we excluded one newly formed keloid sample from GSE92566 for better data consistency). The GSE7890 microarray dataset [(HG-U133 Plus 2) Affymetrix Human Genome U133 + 2.0 array], which contains 10 samples, was also annotated on the GPL570 platform (five keloid, five normal skin).
Affymetrix human genome u133 plus 2
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Affymetrix Human Genome U133 Plus 2.0 is a gene expression microarray platform. It is designed to analyze the expression level of over 47,000 transcripts and variants, representing approximately 38,500 well-characterized human genes.
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5 protocols using affymetrix human genome u133 plus 2
1
Comparative Analysis of Keloid Transcriptomes
2
Reannotation of Affymetrix U133 Plus 2.0 Probes
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Reannotation of the probe IDs for Affymetrix Human Genome U133 Plus 2.0 (Affymetrix; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was required to obtain data regarding lncRNA expression. Initially, we downloaded the HG-U133 Plus 2 annotation file from Affymetrix (CVS format; edition no. 36; www.affymetrix.com/estore/index.jsp ; Affymetrix; Thermo Fisher Scientific, Inc.), which included 54,674 probe IDs for coding RNAs, microRNAs, lncRNAs and other non-coding RNAs. The criteria for assigning lncRNA probe IDs were as follows: i) The Refseq ID started with NR_ or XR_, indicative of non-coding RNAs; ii) the Ensemble gene IDs' annotations were antisense, processed transcripts, sense overlapping, non-sense mediated decay, sense intronic or lincRNA; and iii) pseudogenes, rRNAs, microRNAs, and other small RNAs including tRNAs, snRNAs and snoRNAs were excluded. Finally, 3,809 probe IDs were reannotated as lncRNAs in Affymetrix Human Genome U133 Plus 2.0 (Affymetrix; Thermo Fisher Scientific, Inc.) representing 2,964 different lncRNAs (17 (link)).
In addition, mRNA probe IDs were identified by excluding non-coding RNAs (lncRNAs, microRNAs, pseudogenes, rRNAs, tRNAs, snRNAs and snoRNAs). Probe IDs encoding more than one mRNA and those without RefSeq Protein IDs were excluded. Finally, 38,429 probe IDs remained, representing 17,510 mRNAs.
In addition, mRNA probe IDs were identified by excluding non-coding RNAs (lncRNAs, microRNAs, pseudogenes, rRNAs, tRNAs, snRNAs and snoRNAs). Probe IDs encoding more than one mRNA and those without RefSeq Protein IDs were excluded. Finally, 38,429 probe IDs remained, representing 17,510 mRNAs.
3
Comparative Analysis of Melanoma Transcriptomes
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GEO (http://www.ncbi.nlm.nih.gov/geo ) (8 (link)) is a publicly available function-related genomics repository containing high-throughput gene expression data, ChIP-seq data, as well as microarrays. Three gene datasets, GSE3189 (9 (link)), GSE4570 (10 (link)) and GSE4587 (11 (link)), were downloaded from the GEO database. GSE3189 and GSE4570 were based on the ArrayGPL96 platform (Affymetrix Human Genome U133A Array), whereas GSE4587 was based on the GPL570 platform (Affymetrix Human Genome U133 Plus 2.0). The probes were later converted to their analogous gene symbols using platform information. The GSE3189 dataset comprised 45 melanoma tissue samples and 18 normal nevi tissue samples, GSE4570 contained 6 melanoma samples and 2 nevi samples, and GSE4587 contained 7 melanoma samples and 8 nevi samples.
4
Analyzing lncRNA Expression in Sepsis
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To explore how the lncRNAs are expressed in sepsis, we reannotated lncRNAs based on three sepsis adult whole blood gene expression datasets, GSE95233, GSE57065, and GSE28750. All of them are on the same platform of Affymetrix Human Genome U133 Plus 2.0 that were designed for detecting the expression intensity of coding genes. The platform of Affymetrix Human Genome U133 Plus 2.0 Array has been widely used for gene expression profiling of patient with sepsis [40 (link), 41 (link)]. On top of this, it has the most comprehensive coverage of the annotated human lncRNAs. Using the latest NetAffx Annotation File, HG-U133_Plus_2 Annotations (Release 35, 04/16/15), we reannotated the lncRNAs of the three datasets as follows: (1) The RefSeq ID labeled with NR_ or XR_, indicative of non-coding RNAs, are retained; (2) the Ensemble gene IDs annotated with antisense, processed transcripts, sense overlapping, non-sense mediated decay, sense intronic or lincRNA are retained; (3) pseudogenes, rRNAs, microRNAs, and other small RNAs including tRNAs, snRNAs and snoRNAs are filtered out. Finally, 5016 probesets were detected as lncRNAs representing 3640 unique lncRNAs. Probesets encoding more than one lncRNA were averaged.
5
Keloid vs. Normal Scar Fibroblast Transcriptomics
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The microarray dataset GSE7890 was obtained from the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo). This dataset mainly includes a keloid broblast group without prednisone treatment (n = 5), a normal scar broblast group without prednisone treatment (n = 5), a keloid broblast group after prednisone treatment (n = 5), and a normal scar broblast group after prednisone treatment (n = 4) 4 . Based on the needs of the experiment, we downloaded the raw data for the keloid and the normal scar groups without prednisone treatment (including the Affymetrix CEL le and the probe annotation le) and performed a further analysis. The platform of the microarray was GPL570
[Affymetrix Human Genome U133 Plus 2.0 (Affymetrix, USA)].
[Affymetrix Human Genome U133 Plus 2.0 (Affymetrix, USA)].
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