Identification of 4370 expressed sequence tags from a 3'-end-specific cDNA library of human skeletal muscle by DNA sequencing and filter hybridization.

Abstract

A systematic study on the mRNA species expressed in the human skeletal muscle is presented in this paper. To carry on this study, a new method has been developed for the construction of unbiased cDNA libraries specially designed for the production of ESTs corresponding to the 3'-end portion of the mRNAs. The method has been applied to human skeletal muscle, where the analysis of the transcription profile is particularly difficult for the presence of several very abundant transcripts. To detect and quantify high-level mRNAs, the first 1054 ESTs were obtained from randomly selected clones. The 10 most abundant transcripts accounted for >45% of the clones. Subsequently, these transcripts were identified by filter hybridization, thus making DNA sequencing more productive. Overall, 4370 clones were identified: 3372 by DNA sequencing and 998 by filter hybridization. The number of groups of sequences identifying individual transcripts was relatively low compared with other tissues, resulting in a total of 934 groups out of 4370 ESTs. Of these, 719 groups were represented by only one sequence.

Gene expression profiling identifies potential relevant genes in alveolar rhabdomyosarcoma pathogenesis and discriminates PAX3‐FKHR positive and negative tumors

“…GPL2011). These libraries were produced using a strategy that allows the selection of a short 3 0 -end region of mRNAs 36 and the tagging of each transcript with an unique probe. Moreover, since the 3 0 -end noncoding region is the least conserved part of the genes, our microarray greatly reduces the cross-hybridization of different mRNAs derived from genes with high similarity in the coding region (e.g.…”

The pattern of gene expression in human CD15+myeloid progenitor cells

“…An important issue for gene identification in the postgenome era is the identification of genes expressed at low levels. The conventional EST approach, or random sequencing of clones from regular cDNA libraries, primarily identifies the genes expressed at high levels, as shown that only thousands of genes can be identified in a library by using this strategy regardless of the large-scale efforts, and most of these genes are already known (23)(24)(25). Applying the subtraction͞normalization strategy can identify more genes expressed at lower levels, many of which are novel genes (5,26).…”

Giant axonal neuropathy locus refinement to a < 590 kb critical interval

“…5,6 Mapping of ESTs and STSs ESTs and STSs sequences were retrieved from public web sites: National Center for Biotechnology Information (NCBI; http:/ /www.ncbi.nlm.nih.gov/genemap98/), Whitehead Institute Center for Biomedical research (http://www-genome.wi.mit.edu/), Généthon (http://www.genethon.fr), Cooperative of Human Linkage Center (CHLC; http://www.chlc.org/) and, for chromosome 16 only sequences, Los Alamos National Laboratory (LANL; http://www-ls.lanl.gov). [9][10][11][12][13][14][15][16][17] Primer sequences, expected PCR product sizes and PCR conditions to amplify parts of these sequences were obtained from these web sites. The ESTs and STSs were PCR amplified from genomic DNA isolated from somatic human-mouse hybrid cell lines that contain a single human chromosome 16 fragment 18 and electrophoresed on sequencing gels.…”