An improved method of sncRNA cDNA library construction

Abstract:

With the increasing recognization of sncRNA, especially miRNA and piRNA, it has been proved that small noncoding RNA (sncRNA，<200 nt) plays a significant role at many levels including gene expression regulation, alternative splicing, cell growth, differentiation, proliferation, apoptosis and disease, etc. The sncRNA cDNA library construction is the main way to acquire and research sncRNA. Now, there are many sncRNA cDNA library kits on the market but it’s too expensive. Therefore, an improved method of sncRNA cDNA library construction was established: (1)5S and 5.8S rRNA were removed efficiently by RNA Antisense Purification (RAP); (2) More 5′-monophosphorylated RNAs were acquired after using Tobacco Acid Pyrophosphatase (TAP) and RNA 5' sncRNA Polyphosphatase, which resulted in the increasement of the coverage of the cDNA library. (3) To avoid using the adenylated adapter, a poly(A) structure was added to sncRNA 3′ end and then reverse transcription was carried out with oligo(dT)16VN-adapter primers. Finally, the complete sncRNA cDNA sequences were obtained by employing this improved and cost-effective method, which laid the foundation for the further study of sncRNA.