Revising RNA Amplification - Improved on-bead amplification of scarce, unknown RNA species

High-throughput sequencing techniques find increasing application to address various biological questions related to RNA, such as the identification of (small) RNAs and RNA-protein interactions, as well as transcriptome analysis [1]. In all cases, it is crucial to efficiently attach primer binding sites to the unknown target sequences.
A number of protocols based on ligase or nucleotidyl transferase mediated addition of primer binding sites exist, but these techniques are often low yielding when target sequences are long or able to form stable secondary structures, which is detrimental for scarce samples. In addition, the enzymes show a severe bias towards terminal nucleotides [2] and/or modifications [3], which in turn causes inhomogeneous data capture during high-throughput sequencing.
We have developed a universal protocol that permits efficient addition of primer binding sites to target sequences, irrespective of length and structural complexity, while reducing the abovementioned sequence bias. This approach suits scarce, chemically lesioned RNA, immobilized on solid support. Difficulties in reverse transcriptase read-through due to chemical lesions, as observed by us and by others [4] are taken into account by carrying out the addition of the 5′ primer binding site at the level of cDNA. We have maximized yields and minimized material losses by combining enzymatic reactions carrying out several of them on solid support, and by eliminating multiple purification steps.