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Due to numerous genome sequencing projects, expression of various protein coding genes, their role in development or disease, and their regulation become more and more deciphered. Recently, the small non-coding miRNAs have been highlighted as major modulators of eukaryotic gene expression. It is suggested that up to 30% of human genes may be under the control of miRNAs. They are involved in essential developmental processes such as timing, embryogenesis, organogenesis, growth control, and programmed cell death; but they also play a role in human disease, in particular cancer.
While the number of human or murine miRNAs increases continuously, there is minor data derived from other species. Hence, there is still a need for convenient and fast cloning methods to obtain sequence data of unknown mature miRNAs from other species beside human or mouse. We have developed a novel method for cloning of small RNA molecules such as miRNAs, which allows simultaneous cloning of up to five small RNA molecules within the same clone. First, RNA molecules < 40 nt are polyadenylated by a poly (A) polymerase. After purification, five different biotinylated 5?-DNA-adaptors are ligated to the molecules in independent reactions. After RT-reactions using oligo-dT-primers with 5?-overhangs and subsequent independent PCRs, the sense strands are purified using Streptavidin-coated magnetic beads. The use of complementary 5?-DNA-adaptors and 5?-overhangs in the different reactions enables the subsequent assembly of the five purified sense strands to one molecule in the following PCR. The products are cloned and isolated plasmids harboring the right insert size are sequenced. Using this method, we have discovered several novel porcine miRNAs.