cDNA Libraries from a Low Amount of Cells

Conventional cDNA library construction often requires a minimum available amount of material (typically 1 or 2 μg of polyA⁺ RNA). For complex organs, such as brain, or certain species, such as humans, as well as subsets of cell types, this condition is often difficult to fulfill. Amplification by polymerase chain reaction (PCR) can be used to circumvent this limitation because it is a powerful method to obtain working quantities of low-abundance DNAs. To effectively apply this method, known sequences need to be attached to the ends of the single-stranded cDNA (ss-cDNA). One at the 5′ end of the ss-cDNA is added during the priming of the synthesis; the other, at the 3′ end, is covalently attached by ligation using the SLIC strategy. With known DNA sequences attached to both ends of the synthetized cDNA, minute quantities can be amplified with sequence-specific primers to provide sufficient material to successfully generate and screen cDNA libraries. The overall scheme is illustrated in Fig. 1 .