Gene Tools announces a new paper describing techniques for miRNA activity knockdown, maturation knockdown and specificity controls using Morpholino oligos
MicroRNAs regulate the expression of genes, often large groups of genes.
They are important in embryonic development, in cardiac function, in carcinogenesis and in many other processes.
The recent discovery of miRNAs has altered our understanding of gene regulation, with many new reports of miRNA activity appearing in the literature.
Experimental manipulation of miRNA activity will become a standard tool of molecular biologists, but currently these techniques are in development and still improving.
MicroRNAs mature through several steps.
A primary miRNA is transcribed from DNA and folds into a stem-loop.
The stem-loop is cleaved from the rest of the transcript by the nucleolytic enzyme Drosha, then the loop is cleaved from the stem by the nucleolytic enzyme Dicer.
The double stranded stem interacts with the protein Argonaute, which cleaves and releases one of the strands, forming the miRISC complex with the other strand, now called the guide strand.
It is the miRISC complex which interacts with mRNAs, altering their expression.
Knockdowns of miRNA activity have generally used oligos targeting the miRNA guide strand.
A Morpholino oligo targeting an miRNA guide strand can interfere with the activity of the miRNA.
It is difficult to control for the specificity of the knockdown when using this technique alone.
However, Morpholinos targeting the nucleolytic processing sites of an immature miRNA can prevent maturation of the miRNA.
This allows sets of nonoverlapping Morpholino oligos targeting a primary miRNA to be used as specificity controls; if two non-overlapping oligos targeting the same miRNA produce the same phenotype, this supports the hypothesis that the phenotype is due to knocking down the activity of the targeted miRNA and not due to an off-target effect.
These techniques are explored in the following paper: Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RHA; Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development; PLoS Biol; 2007;5(8): e203.
