RGD Reference Report - Normalization and subtraction: two approaches to facilitate gene discovery. - Rat Genome Database

Send us a Message
Submit Data |  Help |  Video Tutorials |  News |  Publications |  Download |  REST API |  Citing RGD |  Contact   
Search RGD Grant Resources Citing RGD About Us Contact Us
Genes Variants Community Projects QTLs Strains Markers Genome Information Ontologies Cell Lines References Download Submit Data
OntoMate (Literature Search) JBrowse (Genome Browser) Synteny Browser (VCMap) Variant Visualizer Multi-Ontology Enrichment (MOET) Gene-Ortholog Location Finder (GOLF) InterViewer (Protein-Protein Interactions) PhenoMiner (Quatitative Phenotypes) Gene Annotator OLGA (Gene List Generator) AllianceMine GViewer (Genome Viewer)
Aging & Age-Related Disease Cancer & Neoplastic Disease Cardiovascular Disease Coronavirus Disease Developmental Disease Diabetes Hematologic Disease Immune & Inflammatory Disease Infectious Disease Liver Disease Neurological Disease Obesity & Metabolic Syndrome Renal Disease Respiratory Disease Sensory Organ Disease
Find Models   new Genetic Models Autism Models Rat PhenoMiner (Quantitative Phenotypes) Chinchilla PhenoMiner Expected Ranges (Quantitative Phenotype) PhenoMiner Term Comparison Hybrid Rat Diversity Panel Phenotypes Phenotypes in Other Animal Models Animal Husbandry Strain Medical Records Phylogenetics Strain Availability Calendar Rats 101 Submissions Photo Archive
Pathways
Rat Community Forum Directory of Rat Laboratories Video Tutorials News RGD Publications RGD Presentations Archive Nomenclature Guidelines Resource Links Laboratory Resources Employment Resources

Normalization and subtraction: two approaches to facilitate gene discovery.

Authors: Bonaldo, MF  Lennon, G  Soares, MB 
Citation: Bonaldo MF, etal., Genome Res 1996 Sep;6(9):791-806.
RGD ID: 69939
Pubmed: PMID:8889548   (View Abstract at PubMed)

Large-scale sequencing of cDNAs randomly picked from libraries has proven to be a very powerful approach to discover (putatively) expressed sequences that, in turn, once mapped, may greatly expedite the process involved in the identification and cloning of human disease genes. However, the integrity of the data and the pace at which novel sequences can be identified depends to a great extent on the cDNA libraries that are used. Because altogether, in a typical cell, the mRNAs of the prevalent and intermediate frequency classes comprise as much as 50-65% of the total mRNA mass, but represent no more than 1000-2000 different mRNAs, redundant identification of mRNAs of these two frequency classes is destined to become overwhelming relatively early in any such random gene discovery programs, thus seriously compromising their cost-effectiveness. With the goal of facilitating such efforts, previously we developed a method to construct directionally cloned normalized cDNA libraries and applied it to generate infant brain (INIB) and fetal liver/spleen (INFLS) libraries, from which a total of 45,192 and 86,088 expressed sequence tags, respectively, have been derived. While improving the representation of the longest cDNAs in our libraries, we developed three additional methods to normalize cDNA libraries and generated over 35 libraries, most of which have been contributed to our integrated Molecular Analysis of Genomes and Their Expression (IMAGE) Consortium and thus distributed widely and used for sequencing and mapping. In an attempt to facilitate the process of gene discovery further, we have also developed a subtractive hybridization approach designed specifically to eliminate (or reduce significantly the representation of) large pools of arrayed and (mostly) sequenced clones from normalized libraries yet to be (or just partly) surveyed. Here we present a detailed description and a comparative analysis of four methods that we developed and used to generate normalize cDNA libraries from human (15), mouse (3), rat (2), as well as the parasite Schistosoma mansoni (1). In addition, we describe the construction and preliminary characterization of a subtracted liver/spleen library (INFLS-SI) that resulted from the elimination (or reduction of representation) of -5000 INFLS-IMAGE clones from the INFLS library.

Objects referenced in this article
Gene ARL8A ADP ribosylation factor like GTPase 8A Homo sapiens
Gene CLEC4F C-type lectin domain family 4 member F Homo sapiens
Gene EXOC6 exocyst complex component 6 Homo sapiens
Gene Adam19 ADAM metallopeptidase domain 19 Rattus norvegicus
Gene Adam22 ADAM metallopeptidase domain 22 Rattus norvegicus
Gene Adam23 ADAM metallopeptidase domain 23 Rattus norvegicus
Gene Adam9 ADAM metallopeptidase domain 9 Rattus norvegicus
Gene Amacr alpha-methylacyl-CoA racemase Rattus norvegicus
Gene Aplp2 amyloid beta precursor like protein 2 Rattus norvegicus
Gene Cct4 chaperonin containing TCP1 subunit 4 Rattus norvegicus
Gene Fen1 flap structure-specific endonuclease 1 Rattus norvegicus
Gene Folr1 folate receptor alpha Rattus norvegicus
Gene Fos Fos proto-oncogene, AP-1 transcription factor subunit Rattus norvegicus
Gene Fxyd3 FXYD domain-containing ion transport regulator 3 Rattus norvegicus
Gene Fxyd7 FXYD domain-containing ion transport regulator 7 Rattus norvegicus
Gene Gimap1 GTPase, IMAP family member 1 Rattus norvegicus
Gene Gimap7 GTPase, IMAP family member 7 Rattus norvegicus
Gene Gimap8 GTPase, IMAP family member 8 Rattus norvegicus
Gene Gpx2 glutathione peroxidase 2 Rattus norvegicus
Gene Hck HCK proto-oncogene, Src family tyrosine kinase Rattus norvegicus
Gene Hmox1 heme oxygenase 1 Rattus norvegicus
Gene Klk1c8 kallikrein 1-related peptidase C8 Rattus norvegicus
Gene Kyat1 kynurenine aminotransferase 1 Rattus norvegicus
Gene Llgl1 LLGL scribble cell polarity complex component 1 Rattus norvegicus
Gene Mrpl19 mitochondrial ribosomal protein L19 Rattus norvegicus
Gene Prodh proline dehydrogenase Rattus norvegicus
Gene Ptpmt1 protein tyrosine phosphatase, mitochondrial 1 Rattus norvegicus
Gene Rpl39 ribosomal protein L39 Rattus norvegicus
Gene Rps15 ribosomal protein S15 Rattus norvegicus
Gene Rps5 ribosomal protein S5 Rattus norvegicus
Gene Slc25a1 solute carrier family 25 member 1 Rattus norvegicus
Gene Sp3 Sp3 transcription factor Rattus norvegicus
Gene Sprn shadow of prion protein Rattus norvegicus
Gene Vkorc1 vitamin K epoxide reductase complex, subunit 1 Rattus norvegicus

Additional Information