Figure 1. The Total RNA Detection system.

A universal RNA adaptor was firstly added to the 3′ ends of all RNA templates. The abundant 18S and 28S ribosome RNAs were then subtracted by using biotinylated ribosomal-specific probes. Small-size RNAs containing the degraded RNA intermediates were removed by size-filtration. The enriched transcripts were converted into double-strand cDNA by using the 3′ end RNA adaptor-based primer. The cDNAs were further digested by NlaIII. The 3′ cDNAs were isolated by using the streptoavidin beads. An adaptor was added to the 5′ ends of the 3′ cDNAs. The 3′ cDNAs were then amplified by PCR using the 5′ adaptor-based sense primer and the 3′ end RNA adaptor-based antisense primer. The amplified 3′ cDNAs were sequenced from the 3′ end by the 454 system. See further details in Materials and Methods.