Cloning microRNAs from mammalian tissues.
MicroRNAs (miRNAs) are ubiquitous regulators of gene expression in plants and animals. Their distinctive structure, as very short RNAs with a 5'-phosphate and 3'-hydroxyl group, has enabled the development of protocols to clone miRNAs. After enrichment of these small molecules by size, serial ligation of adapter oligonucleotides to each terminus allows amplification using reverse transcription (RT)-polymerase chain reaction (PCR). Plasmid cloning of multiple miRNA sequences and subsequent DNA sequence analysis enable both bioinformatic characterization of the various miRNAs and experimental validation of their accumulation in cells.
Michael
Michael Z
MZ
Department of Gastroenterology and Hepatology, Flinders Medical Center, Flinders University School of Medicine, Bedford Park, Australia.
D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D019295:Computational Biology; D018076:DNA, Complementary; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D035683:MicroRNAs / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D035683:MicroRNAs