Concatameric cloning of porcine microRNA molecules after assembly PCR

Biochemical and Biophysical Research Communications
Soroush Sharbati-TehraniRalf Einspanier

Abstract

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.

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Citations

Jul 5, 2013·Animal : an International Journal of Animal Bioscience·X WangH Jiang
Feb 7, 2009·BMC Genomics·Alavala Matta ReddyRamanjulu Sunkar
May 4, 2010·BMC Genomics·Soroush SharbatiRalf Einspanier
Jul 17, 2010·PloS One·Mingzhou LiXuewei Li
Feb 12, 2011·Journal of Cellular Biochemistry·Guoxi LiGongshe Yang
Mar 25, 2010·Animal Genetics·B ZhouJ Y Wang
Jul 6, 2010·Animal Science Journal = Nihon Chikusan Gakkaihō·Bo Zhou, Hong-Lin Liu
Nov 25, 2011·Animal Science Journal = Nihon Chikusan Gakkaihō·Yiren GuXuewei Li
Feb 25, 2010·Molecular Biology Reports·Ik Sang ChoYoung Sik Lee

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