Publikationsdatenbank

Concatameric cloning of porcine microRNA molecules after assembly PCR (2008)

Art

Zeitschriftenartikel / wissenschaftlicher Beitrag

Autoren

Sharbati-Tehrani, Soroush

Kutz-Lohroff, Barbara

Scholven, Jutta

Einspanier, Ralf

Quelle

Biochemical and biophysical research communications

Bandzählung: 375

Heftzählung: 3

Seiten: 484 – 489

ISSN: 0006-291x

Sprache

Englisch

Verweise

DOI: 10.1016/j.bbrc.2008.08.048

Pubmed: 18722348

Kontakt

Institut für Veterinär-Biochemie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62225
biochemie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

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.