Single-Cell cDNA Library Construction Using Cycling aRNA Amplification

Abstract

Several existing methods for constructing complementary DNA (cDNA) libraries require several thousand cells and involve the lengthy procedure of reverse transcription (RT), restriction, adaptor ligation, and vector cloning, which usually fail to maintain the completeness of a cDNA library, resulting in a loss of rare cDNAs. However, gene expression analysis of specific cell populations within a heterogeneous tissue is essential for research in vivo, requiring a method to generate cDNA libraries from a very small number of specific cells. The generation of amplified antisense RNA (aRNA) by incorporating an oligo-(dT) primer coupled to a T7 RNA polymerase promoter sequence during RT has been developed to linearly increase transcriptional copies of mRNAs from a limited amount of promoter-linked cDNAs (1,2) (see Note 1). However, the aRNAs prepared from a single live neuron has been reported to cover 50–75% of the total mRNA population (2,3), indicating that rare mRNAs were lost during the amplification procedure.