Concatameric cloning of porcine microRNA molecules after assembly PCR.

While the number of human or murine microRNAs (miRNAs) increases continuously, there are limited data available from other species. We report a novel identification method of small RNAs such as miRNAs, which allows simultaneous cloning of five RNA molecules within the same insert. First, RNA molecules <40nt were polyadenylated and five concatamerising 5' DNA adaptors were ligated to the molecules in independent reactions. Reverse transcription was carried out using oligo d(T)(18) primers with concatamerising 5' overhangs. The introduced complementary termini in the different reactions enabled the subsequent coupling of five purified antisense strands to one molecule by means of an assembly PCR. After cloning, small RNAs were identified by DNA sequencing. By means of this cloning approach, we identified 10 novel and one known porcine miRNAs. Furthermore, the endogenous expression of the cloned miRNAs was quantified in various tissues using a qRT-PCR approach.