A Risky Pi

Ines Fournon Berodia

Our researcher from Stazione Zoologica Anton Dohrn, SZN in Naples

Konstantinos Geles

Our researcher from ESR, Università degli Studi di Salerno, UNISA

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A Risky Pi

How much work would you put into something that may not be of any significance?

Baking

They say baking is a science.

It‘s all about accuracy in its methods and
results that can be reproduced consistently.

If the instructions are followed precisely,
the end product is always the same.

Protocols

Biologists follow recipes too,
only they call them 'Protocols'.

With almost no over-the-counter ingredients and molecular level precision required, protocols can sometimes take years to figure out.

Protocols

Biologists follow recipes too,
only they call them 'Protocols'.

With almost no over-the-counter ingredients and molecular level precision required, protocols can sometimes take years to figure out.


And then the data they generate might not even give them the answers they are looking for.

But that’s science!

Our Researchers

Inés & Constantinos are interested in the role of piRNAs.

These tiny molecules that exist in all cells ... not much is known about them.

Risks

This adds another layer of risks -we know little about this molecule, it might not be of any significance.

Risks

This adds another layer of risks -we know little about this molecule, it might not be of any significance.


Or...it might become the next big
superstar of molecules?

Risks

This adds another layer of risks -we know little about this molecule, it might not be of any significance.

Or...it might become the next big
superstar of molecules?


Like mRNA in 2021.

Let's follow Inés‘ research where she wants to understand the role of piRNA in passing down the genetic information to the future generation of sea urchins.

Hence, she is looking for a very specific recipe:
 
How to extract the piRNAs of germ cells
in sea urchins?

Just like finding a good recipe online,
biologists too rake through research
papers to find a protocol that best
suits their needs.

After a long search,
Ines found a promising recipe -
a research paper by Swartz et. al.
from 2014.

She used the first half of the
protocol and tweaked it a little.

Let's follow Ines in her experiment
to extract the piRNAs.

She starts with placing the sea urchin egg in a petri dish and then fertilises it with sperm.

The fertilised egg is called a zygote.

Once the zygote has divided itself into 16 cells, two germ line cells appear.

Cross section view

Once the zygote has divided itself into 16 cells, two germ line cells appear.


These are responsible for passing down the genetic information to the future generation.

Cross section view

Inés waits 15 hours after fertilization.

She should now have about 200 cells.

Cross section view

She now uses a green molecular dye called “calcein AM” to label the four germline cells.

Image of stained cells by Ines Fournon Berodia (CC BY-NC-ND)
Real Image
Cross section view

In the next step, she splits the group of cells into many single cells.

Once split, she has a mix of labelled and unlabelled cells floating around alive in a liquid that allows the cells to be "happy".

Without the right balance of salts the cells would explode!

Image of dissociated cells by Ines Fournon Berodia (CC BY-NC-ND)
Real Image

She then places the mix of single
cells into a tube.

To separate labelled from unlabelled cells, she connects the tube to a machine called Fluorescent Activated Cell Sorter, in short, the FACS machine.

The machine will pass the cells through a laser that will recognise the labelled cells and place them in a separate tube.

She then needs to extract the RNA (which also contains the piRNA) from the labelled cells.

For this she follows another protocol that uses a range of different chemicals that allow her to separate the RNA from the DNA and proteins.

The chemicals separate the RNA, which is now suspended in the pink layer.

The chemicals separate the RNA, which is now suspended in the pink layer.


She puts them into another test tube.

She then transports the RNA in dry ice to the southern corner of Italy, Salerno, where Konstantinos works at Genomix4Life.

There, Konstantinos places the RNA into a machine that translates the RNA into readable codes of “A” “U” “C” “G”.

He ends up with a massive amount of code, called RNA sequences.

As a last step Konstantinos
filters out the code for the piRNAs,
as there are many types of RNAs.

For this he uses 3 types of filters.

The length filter.

The pattern filter.

The expression filter.

And finally we end up with the piRNA sequences we were looking for.

And what now?

And what now?


This is when the second part
of the research begins:
analyzing the data.

And what now?

This is when the second part
of the research begins:
analyzing the data.


Do piRNAs play an important role in
passing down genetic information to
the next sea urchin generation?

And what now?

This is when the second part
of the research begins:
analyzing the data.

Do piRNAs play an important role in
passing down genetic information to
the next sea urchin generation?


Are they important at all?

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