An improved method for constructing a full-length enriched cDNA library using small amounts of total RNA as a starting material

Abstract

We have developed an improved method for constructing a full-length cDNA library using small quantity of material by modifying the original oligo-capping method. In our devised method, total RNAs are used in sequential oligo-capping steps directly without preliminary mRNA purification. Using this method, we constructed full-length cDNA libraries from 100 μg of total RNA. These libraries contained 8 × 105 to 8 × 106 independent clones with average insert sizes of 2.0 kb. Moreover, the number of full-length cDNAs containing the translation initiation codon ATG in the constructed libraries was estimated to 60-70%. In addition, 54% of the known cDNAs had a longer 5′ end than the corresponding genes in the public database. Our results show that the method can be effectively used to construct full-length enriched cDNA libraries, especially, if starting material is limited.