How can I be sure we are targeting only miRNA in the total-RNA?
This is achieved by two-step discrimination (1) sequence hybridization – the specificity of the miRNA specific probe which targets the pre and mature miRNA species, and (2) enzymatic primer extension – to enhance the discrimination between members of miRNA families and between miRNA and other similar sequences in the total-RNA (e.g., mRNA targets). Illumina has obtained very similar expression profiles with total-RNA and enriched small RNA species, suggesting that cross-hybridization (if any) from the total-RNA is minimal.
