Putting Micro RNAs Under the Microscope

Posted by Neil Emans
On Feb 10, 2016

The human genome comprises around twenty thousand genes, which feels like a significant number. However, the maize plant that grows the corn that eventually makes cinema popcorn has at least forty three thousand which is a sobering observation. Even the nematode has almost as many genes as Leonardo Da Vinci.

Our genome is 4 billion base pairs long, yet it contains vast stretches of DNA that do not encode proteins, compared to the parts that do. For many years this space was called “junk” DNA, because little function could be assigned to it. It lies between the expressed pieces of genes: exons. More than 95% of the genome is this space. Between the exons, that are stitched together to form complete genes, the spaces were a mystery. Until recently, this was essentially in the shadows, the genome’s dark matter

All that changed in the past 25 years with the discovery of RNA molecules that are encoded in the dark matter. These have had various names, the most accurate “non-coding RNA”. Micro-RNAs are a sub-set of these RNAs and are found in plants, animals, viruses and us.

Practically unknown until 25 years ago, micro-RNAs are emerging as a focus of research. Micro-RNAs are molecules our cells synthesize and that control a wide number of biological process, from cell proliferation to cancer development and even infectious disease. They are a family of a few thousand molecules but they affect close to two thirds of the genes we have

We performed a miRNA screen to identify those that affect RELA expression, using the Persomics platform where the miRNA were printed, in a method very close to siRNA printing. The miRNA arrays were overlaid with Hela cells for 48h, to allow miRNA transfection and action, then fixed and RELA was detected by indirect immuno-labelling with an anti-RELA primary and a green fluorescent secondary. 

One of the miRNA we identified was HSA MIR 373 3P which is a known regulator of RELA expression. Images of miRNA silencing/repression are shown below.

RNAIMAX HSA MIR 373 3P

RNAIMAX-HSA-MIR-373-3P
This microRNA was identifed and the above figure shows six spots containing it (left panel red) after HeLa cell overlay for 48h and staining as described above. The right panel shows the three color image of the spot (acquired on the BioTek Cytation 3) and in the right panel we have turned off the red channel to better show the silenced cells over the spot. 

micro-RNA CONTROL

micro-RNA-control

The micro-RNA CONTROL gives no phenotype related to RELA, as shown above. Quanatitative image analysis showed the repression of RELA by HSA MIR 373 3p to be around 50% - which is often observed for miRNA. This screen was fascinating and we look forward to validating more High Content Biology on the miRNA arrays.

Looking down the microscope at micro-RNA 

At Persomics, we are actively working on extending our platform to encompass more than RNA- interference, and watch this space for more developments as they literally roll off our printing press.

Want to see pictures in higher resolution? Click below to download PDF.


Topics: Science, Updates

Written by Neil Emans

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