Week 8: Truth

It's finally coming together.

So I finally found out answers to a TON of my questions - the most important one being, how is all this molecular bio stuff related to the neuroanatomy side I was working on about half a month ago?

It all starts with the Rdl subunit. The inhibitory GABAa receptor in the honey bee has subunits, and they work together to get the receptor to work. Ligand-gated ion channels are found in all these subunits, and all the subunits should be important to signaling. The Rdl subunit is one such subunit.

When the lab did the genetic and molecular biology side of this experiment (which is still ongoing, I did another round of RNA Isolation this week), the lab injected RNA into the bees to affect expression of the proteins needed to make the Rdl subunit. There were three treatments - dsiRNA (Dicer-substrate short interfering RNA), scrambled RNA, and non-injected. The dsiRNA specifically interferes with the sequence in question, called AmelRdl, that codes for the receptor proteins, preventing its transcription. Scrambled RNA is just nonsense RNA that shouldn't have any effect on expression. And the non-injected group just didn't get any injections at all, so it's a control group.

So the injections happened, and then, the actual experiment commenced (Note: These injections are completely different from the injections I did during the neuroanatomical experiment - that time, I was injecting neurobiotin dye for imaging). All three groups were subject to PER (Proboscis Extension Reflex) conditioning experiments, and then the bees were analyzed, via RNA Isolation and RTq-PCR, to check for levels of the AmelRdl RNA sequence in question after conditioning. This sequence codes for the receptor proteins in question, so correlating levels of this RNA sequence to observed behavior can give us conclusions about the role of the Rdl proteins, and thus, the subunit.

This brings me to the answer to another question I had: Why RNA?

If we do regular PCR on the bees, we get amplification of the bee's entire genome, regardless of whether parts of the genome are being expressed or not. The experiment cares about tracking expression of the right sequence, which would mean we only care about the parts of the genome being expressed. That's why we collect RNA - RNA is present when those corresponding parts of the genome are being expressed. And in the collected RNA, there should be the AmelRdl RNA sequence.

So now, the connection with the neuroanatomical side. I looked at our poster again today, and looking at the conclusions at the bottom of the poster, I noticed that it said the AmelRdl receptors aren't found in the olfactory receptor neurons.

The conclusion I said from the neuroanatomical experiment about the GABAa receptors and the olfactory receptor neurons was basically the TL;DR version of the more specific conclusion - the Rdl subunit isn't found in those neurons. So the entire experiment is not only based around a specific receptor, but is based around a specific subunit in it.

So where am I now in this experiment? Like I stated above, this week, I did another round of RNA Isolation, this time, for the GABAa receptor experiment. Mary's experiment concluded last week (refer back to last week's post if you don't know who I'm talking about), so now I'm doing this procedure for my experiment. Giulia (the other undergrad in my project) provides the samples, and I analyze them. Next week, Dr. Sinakevitch said I'd be doing the RTq-PCR process alone for my samples. This should be pretty fun - will get tons of pics. She'll probably have me practice micropipetting again before I start though - micropipetting for RTq-PCR is extremely precise - I have to micropipette a drop of about 2 microliters onto the side of a sample well about 2 millimeters in diameter. Not even on the top. Bummer - another morning of dull practice.

And...I just remembered about the images over the last few weeks. I will get them up right now.