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Molecular markers for cell types of the inner ear and candidate genes for hearing disorders.

ABSTRACT: To identify genes expressed in the vertebrate inner ear, we have established an assay that allows rapid analysis of the differential expression pattern of mRNAs derived from an auditory epithelium-specific cDNA library. We performed subtractive hybridization to create an enriched probe, which then was used to screen the cDNA library. After digoxigenin-labeled antisense cRNAs had been transcribed from hybridization-positive clones, we conducted in situ hybridization on slides bearing cryosections of late embryonic chicken heads, bodies, and cochleae. One hundred and twenty of the 196 clones analyzed encode 12 proteins whose mRNAs are specifically or highly expressed in the chicken's inner ear; the remainder encode proteins that occur more widely. We identified proteins that have been described previously as expressed in the inner ear, such as beta-tectorin, calbindin, and type II collagen. A second group of proteins abundant in the inner ear includes five additional types of collagens. A third group, including Coch-5B2 and an ear-specific connexin, comprises proteins whose human equivalents are candidates to account for hearing disorders. This group also includes proteins expressed in two unique cell types of the inner ear, homogene cells and cells of the tegmentum vasculosum.

SUBMITTER: Heller S

PROVIDER: S-EPMC21654 | biostudies-literature | 1998 Sep

REPOSITORIES: biostudies-literature

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Molecular markers for cell types of the inner ear and candidate genes for hearing disorders.

Heller S S Sheane C A CA Javed Z Z Hudspeth A J AJ

Proceedings of the National Academy of Sciences of the United States of America 19980901 19

To identify genes expressed in the vertebrate inner ear, we have established an assay that allows rapid analysis of the differential expression pattern of mRNAs derived from an auditory epithelium-specific cDNA library. We performed subtractive hybridization to create an enriched probe, which then was used to screen the cDNA library. After digoxigenin-labeled antisense cRNAs had been transcribed from hybridization-positive clones, we conducted in situ hybridization on slides bearing cryosections ...[more]

PMID: 9736748

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Project description:Purpose: To identify orthologous genes in Xenopus that are implicated in deafness and vestibular disorders in humans and to compare RNA-Seq and microarray to determine the advantages of each technology for Xenopus transcriptomic analysis. Methods: Inner ear RNA from X. laevis stages 56-58 was isolated with the QiagenÂ® RNeasyÂ® Mini Kit. RNA quality was assessed using the Agilent 2100 Bioanalyzer. The RNA was sent to the National Center for Genome Resources, for Illumina-Solexa sequencing using the genome analyzer II and to MIT for microarray processing usign the Affymetrix GeneChipÂ® X. laevis Genome 2.0 Array. The OMIM database was mined for identification of genes causing deafness and vestibular disorder in humans. Human sequences for each of the deafness and vestibular disorders genes were downloaded from Ensembl. Blast-2.2.27+ was used to mapped the human sequences against X. tropicalis predicted proteins from JGI or to Xenopus consensus sequences downloaded from Affymetrix. The alignment of the human sequences to the predicted proteins resulted in the identification of the protein designation number. The protein designation number was used to obtain the sacaffold number and coordinates from JGI genome browser. The scaffold number and coordinates were input into Alpheus sequence variant detection pipeline to obtain the number of reads associated with each gene. The reads were log2 transformed for comparison to the microarray. In the case of the alignment of the human sequences to the Xenopus consensus sequences resulted in the PSID. The PSID was used to obtain the GCRMA intensity value. The GCRMA value and the number of reads were compared to determine which technology detected more genes. Results: Using Affymetrix GeneChipÂ® X. laevis Genome 2.0 Arrays and Illumina-Solexa sequencing methods, we determined that the transcriptional profile of the Xenopus inner ear comprises hundreds of genes that are orthologous to OMIMÂ® genes implicated in deafness and vestibular disorders in humans. Analysis of genes that mapped to both technologies showed that RNA-Seq detected more of both deafness and vestibular disorder than the Microarray. RNA-Seq and microarray detected 108 genes but RNA-Seq detected 48 genes that were below threshold criteria for microarray. While the microarray detected 11 genes that were below the expression criteria for RNA-Seq and 23 genes were below the threshold criteria for both technologies. Further analysis of the 108 genes detected by both technologies showed a minor correlation between the two technologies. Conclusions: As part of this study we identified candidate scaffold regions of the Xenopus tropicalis genome that can be used for gene mutagenesis. Furthermore, results from this study showed that the two technologies can complement each other and that a combination of both approaches can provide a more complete analysis of gene expression than either method alone. Inner ear RNA from X. laevis larval stages 56-58 was isolated and shipped to the National Center for Genome Resources, for Illumina-Solexa sequencing or to the Massachusetts Institute of Technology BioMicro Center for microarray analysis with the Affymetrix GeneChipÂ® X. laevis Genome 2.0 Array. RNA-Sequencing was completed using the Illumina-Solexa platform for sequencing by synthesis. Short-insert paired end (SIPE) libraries were prepared from total RNA according to Illuminaâ??s mRNA-Seq Sample Prep Protocol v2.0 (Illumina, San Diego, CA, USA). The resultant double-stranded cDNA concentration was measured on a NanoDrop spectrophotometer, and size and purity were determined on the 2100 Bioanalyzer using a DNA 1000 Nano kit. The cDNA libraries were cluster amplified on Illumina flowcells, sequenced on the GAII Sequencer as 36-cycle single-end reads, and processed using Illumina software v1.0. Illumina reads were aligned to the X. tropicalis genome using the algorithm for genomic mapping and alignment program (GMAP) and AlpheusÂ® Sequence Variant Detection System v3.1.

Project description:Purpose: To identify orthologous genes in Xenopus that are implicated in deafness and vestibular disorders in humans and to compare RNA-Seq and microarray to determine the advantages of each technology for Xenopus transcriptomic analysis. Methods: Inner ear RNA from X. laevis stages 56-58 was isolated with the Qiagen® RNeasy® Mini Kit. RNA quality was assessed using the Agilent 2100 Bioanalyzer. The RNA was sent to the National Center for Genome Resources, for Illumina-Solexa sequencing using the genome analyzer II and to MIT for microarray processing usign the Affymetrix GeneChip® X. laevis Genome 2.0 Array. The OMIM database was mined for identification of genes causing deafness and vestibular disorder in humans. Human sequences for each of the deafness and vestibular disorders genes were downloaded from Ensembl. Blast-2.2.27+ was used to mapped the human sequences against X. tropicalis predicted proteins from JGI or to Xenopus consensus sequences downloaded from Affymetrix. The alignment of the human sequences to the predicted proteins resulted in the identification of the protein designation number. The protein designation number was used to obtain the sacaffold number and coordinates from JGI genome browser. The scaffold number and coordinates were input into Alpheus sequence variant detection pipeline to obtain the number of reads associated with each gene. The reads were log2 transformed for comparison to the microarray. In the case of the alignment of the human sequences to the Xenopus consensus sequences resulted in the PSID. The PSID was used to obtain the GCRMA intensity value. The GCRMA value and the number of reads were compared to determine which technology detected more genes. Results: Using Affymetrix GeneChip® X. laevis Genome 2.0 Arrays and Illumina-Solexa sequencing methods, we determined that the transcriptional profile of the Xenopus inner ear comprises hundreds of genes that are orthologous to OMIM® genes implicated in deafness and vestibular disorders in humans. Analysis of genes that mapped to both technologies showed that RNA-Seq detected more of both deafness and vestibular disorder than the Microarray. RNA-Seq and microarray detected 108 genes but RNA-Seq detected 48 genes that were below threshold criteria for microarray. While the microarray detected 11 genes that were below the expression criteria for RNA-Seq and 23 genes were below the threshold criteria for both technologies. Further analysis of the 108 genes detected by both technologies showed a minor correlation between the two technologies. Conclusions: As part of this study we identified candidate scaffold regions of the Xenopus tropicalis genome that can be used for gene mutagenesis. Furthermore, results from this study showed that the two technologies can complement each other and that a combination of both approaches can provide a more complete analysis of gene expression than either method alone.

Dataset Information

Molecular markers for cell types of the inner ear and candidate genes for hearing disorders.

Publications

Molecular markers for cell types of the inner ear and candidate genes for hearing disorders.

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