A novel ligation mediated-PCR based strategy for construction of subtraction libraries from limiting amounts of mRNA

摘要

Construction of subtraction libraries has become a popular method for isolating differentially activated genes. Several methods have already been published, but all of them require either ≥10 μg of polyA~+ RNA (1), a large amount of total RNA (2) ora pre-made cDNA library (3-7). This could be a problem when dealing with expensive or scarce cell lines or tissues. The recent development of the 'differential display' technique (8) represents one means of overcoming the requirement for a significant amount of RNA, but some differentially expressed transcripts may not be isolated by this procedure because of peculiarities in their sequences. Here I present a ligation mediated-polymerase chain reaction based subtraction strategy which is quick, efficient, moderately expensive, and requires ≤0.5 μg polyA~+ RNA.