Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hybridization system

Abstract

We report a novel procedure to construct a normalized (equalized) cDNA library. By introduction of the highly efficient self-hybridization system between a whole mRNA population and their corresponding cDNA immobilized on latex beads, which involves relatively simple manipulations, we were able to generate an mRNA population in which the copy number of abundant species was reduced while that of rare species was enriched. In a typical experiment, after several cycles of self-hybridization on the beads, the ratio of the most to the least abundant marker mRNA species dropped by a factor of 300 (from 10,000 to 30) while the complexity and length of mRNAs in the population remained unchanged. The procedure should provide a potent tool for the expression cloning of cDNA and also facilitate the construction of whole cDNA catalogs from specific tissues (or cell types) from higher organisms.

Expression of the human cGMP‐dependent protein kinase II gene is lost upon introduction of SV40 T antigen or immortalization in human cells

“…To isolate a full-length cDNA clone, the above sequence was labeled with ["P]dCTP by random priming and used to screen approximately 1 x 10" recombinant 1ZapII phage clones containing cDNA prepared from TIG3 cells using a cDNA synthesis kit (BRL-choice &stem) as described [18,19]. The phage clones were converted to pBluescript in vivo and the inserts were subcloned to sequence both strands [20].…”

Initial gene expression profile of rainbow trout (oncorhynchus my kiss)intestine

“…If the EST analysis is for the purpose of EST cataloguing for development of bio-component, repeated sequencing of highly expressed genes is not desirable. Normalized cDNA libraries (Patanjali et al, 1991;Sasaki et al, 1994) or subtracted cDNA libraries are therefore needed for characterization of large numbers of unique ESTs. Subtracted cDNA libraries of rainbow trout intestine were developed in our laboratory using highly expressed genes whose cDNA clones were found more than once as subtractants.…”

Expression Pattern in a Modified Equalized Kidney cDNA Library of Hypertensive Rat

“…The efficiency of equalization/normalization procedures in reducing the numbers of abundant species of transcript in an ordinary cDNA library has been shown by pilot investigations [9, 13, 14, 15]. However, systematic comparison between the expression patterns of an equalized/normalized cDNA library and that of an ordinary cDNA library is rarely reported.…”

“…These cDNA fragments are probably the most specific sequences of their corresponding transcripts. Because the sequences of 3′ untranslated regions were thought to be specific for their transcripts, 3′ end cDNA fragments were used for equalization/normalization procedures in most studies in order to preserve the complexity of the cDNA libraries [9, 14, 15]. In theory, if the sequences of 3′ end regions were really specific for their transcripts, there would be a high proportions of 3′ end fragments remaining after equalization/normalization.…”