RT-PCR in cDNA Library Construction

The construction of high-quality, full-length cDNA libraries is still the ideal method for cloning and studying mRNA populations. However, a significant problem with conventional cDNA library construction is the requirement for microgram quantities of poly A⁺ RNA at the start of the procedure (1 ). If the amount of biological material is limited and resultant poly A⁺ RNA yields are low (<500 ng), the conventional approach is not an option. This problem will arise, for example, if one is attempting to construct cDNA libraries from a small number of cells, or indeed a single cell, from a tissue with a large number of cellular phenotypes such as the brain. Secondly, the investigation of gene expression at various time-points during embryonic development can be facilitated by the construction of cDNA libraries, but in many cases the amount of available embryonic tissue is minute. One obvious solution to this problem is to increase the amount of starting material, but this approach could result in higher costs and potential ethical difficulties associated with sacrificing large numbers of animals or obtaining samples from a greater number of volunteers or patients.