Paul's Results

Hey All,

Just wanted to let you know that the results are in!
We have observed many genes that play a role in heat response.
The most interesting genes are the ones that also play a role in photosynthesis. This infers that the response to light stimuli and the response to heat shock follow a rather similar pathway.

This light response/heat shock response pathway was inferred by several articles in the scientific literature. The fact that we were able to repeat previous findings gives validation to both our experimental results and to the implication that the two stimuli are connected.

Pretty exciting, eh??

Field Trip!!!!

The moment we've been waiting for has finally come!!!!

We took a field trip to the microaray facilities at the University of Pennsylvania in order to scan our microarray slides. We all jumped into a van and Karl safely drove us (thanks Karl!). When we got there, we met Ben, who helped us with our scanning.

What's the whole scanning process all about?

Well, be patient and we'll tell you.

Basically, the microarray slide with our hybridized sample is exposed to UV light. This exposure causes the Cy3 to glow a green color and Cy5 a red color. When cy3 and cy5 overlap, they glow a yellow color.

So remember we labeled our control population cDNA with the cy5 dye and our treated population cDNA with a cy3 dye. Also remember that cDNA is a product of RNA so it is indicative of whether a gene is expressed or not. Also remember that the microarray slide has many spots on them, to which different cDNAs corresponding to different genes can bind.

Why do you have to remember so many things??

The answer is so that you will understand that a red signal for a certain spot on the microarray slide (which corresponds to a certain gene) means that the particular gene is expressed in the control population and not expressed in the heat shock treated group - hence, one might suspect that this gene is turned off during heat shock.

On the other hand, a green signal means that the particular gene is turned on during heat shock, and a yellow signal means that the gene is on in both populations.

By determining which genes are on and off, we can reveal 'heat shock' genes - that is, genes that are involved with the heat shock response.

So what would 25 Haverford College Biology majors be doing in a dimly lit laboratory right before Thanksgiving break? Synthesizing cDNA and creating labeled targets, of course!

The goal for this week was to transcribe cDNA from our RNA samples. But when we transcribed our labeled targets, we wanted to be able to distinguish one sample from the other. Therefore, we used two different cyanine fluorescent dyes as part of our cDNA synthesis to help us visualize our unique samples on the microarray.

But why were we working in the dark? (There was a reason for the madness). The cyanine dyes that we are using are sensitive to light and begin to degrade when exposed to natural or fluorescent light. And so, we wanted to use minimal lighting to synthesize our cDNA using cyanine labeled dNTPs.
dNTPs are dinucleotide triphosphates which provide the bases when transcribing cDNA from RNA. Each dye was specific to a sample so that we would be able to distinguish one sample from the other.

So for our circadian rhythm group, the Cy3 labeled samples will light up red for genes that are expressed in the light period and the Cy5 labeled samples will light up green for genes that are expressed in the dark period. For genes that are expressed in both conditions, we will see a yellow dot.

Ensuring Good RNA Quality

So we've isolated our RNA and now we're going nuts to ensure its quality is acceptable for microarray analysis.

Well, maybe we're being harsh when we say we're going nuts - it is really important to ensure that our RNA is not degraded.

We're doing this using electrophoresis technique. This ensues forcing our product through a network of cross linked polymers (referred to as the "gel").
RNA is negatively charged so we can pull it through the gel using its electromagnetic attraction to positive charge (and that's exactly what we do!).

Smaller, negatively charged fragments move farther down the gel while larger fragments end up higher on the gel. In the end, we can see the different components of our product by observing all the fragments at different positions on the gel.

This technique will allow us to make sure our RNA isn't degraded. Good RNA will show us a certain set of bands (2 bands with a smear above) while degraded RNA will show us the same pattern but with an additional band at the bottom.
Can you guess what that small fragment at the bottom is?
It's degraded RNA - we're crossing our fingers that we don't see it (wish us luck!).

RNA Extraction

Hey y'all - it's Paul speaking.

I'm in lab right now, and we're extracting RNA from 2 populations of chlamydomonas (algae) cells: one was exposed to a heat shock and the other just enjoyed the normal room temperature environment.
Our aim is to investigate and contrast the RNAs transcribed during the normal environment and during the heat shock.

By looking at the different RNAs, which is directly reflective of DNA, we can deduce which genes are involved during the heat shock response.

So what's the similarity between mean old mob bosses and superlab students?
We both use chloroform (but in different ways, of course!)
Mob bosses put chloroform on a towel and shove it into thier victims' faces to knock them out.
We use chloroform to isolate RNA from other proteins in the chlamydomonas cell.

Afterwards, we'll remove the liquid layer contain our RNA from the liquid layer containing the cellular proteins (this ensues a lot of skilled hand work with the pipette). Then, we'll precipitate the RNA from the solution with ethanol, degrade any DNA that's in solution, and then wash our RNA precipitate to ensure good quality.

Stay tuned to see what we do with out RNA!!!

Now that we've shown you how much fun superlab can be, we'll get down to business on the academics...

As Paul mentioned earlier, his group is looking RNA expression levels of chlamydomonas in response to heat shock. Some other topics that groups are looking at are: the effects of nutrient deprivation, bacterial defense mechanisms, and drug inhibition response. So each group is exploring something completely different! At the same time, our professors give us tons of support and guidance along the way in manipulating our protocols. We are also using journal articles to help us design our experiments provide us with some background knowledge on what results we might expect.

My group is interested in the process of circadian rhythms in chlamydomonas. Since such a variety of different experiments involving circadian rhythm have been done, we wanted to narrow down our search to a more specific question that interested us: What are some of the differences in RNA expression level of chlamydomonas in the dark period verus the light period? Since we know certain genes that are involved in photosynthesis are transcribed during the light cycle, we thought we could compare these expression levels with other genes that are involved in processes initiated in the dark cycle, such as cell division! (and no we're not talking about chlamy math.... sorry I couldn't hold back- all Paul's influence!)

So far, we've harvested our cells at both a light stage and dark stage. We then used a solution called trisol, which causes the cell membranes to burst and spill out all of contents of our chlamy cells. Once we had our trisol solution containing our precious RNA along with some DNA and proteins, we wanted to extract just the RNA to see which genes are being transcribed during each stage. More exciting updates to come!

Hey everybody!

This is Paul speaking. First of all, I resent what Jess said/implied about me or my jokes. For the the record, my jokes are bad (I admit it) but neither my jokes or anything about me is nerdy or dorky! I invest a lot in trying to maintain a cool image! (that was a joke) :-)
With that said, let's move on.

Today was superawesome (yes, superawesome is a word. It means totally wicked). Anyway, today in the lab began with a little distraction when Rob, our former superlab professor, came in with a gothic hairstyle just to see people's reaction. The best reaction was from Matt (our current superlab professor along with Karl). You could see the "oh my lord" expression on his face!
While Rob claims that he was dared to come in with that hair style, we all really know that he did everything out of his own free will and the whole dare story is just an excuse.

After the little distraction, we got down to business. My experiment consisted of subjecting one chlamy population to room temperature (as a control) while exposing another to a heat shock. After the exposures, we spliced the cells and froze the contents for RNA extraction which will occur at some later point.

Fortunately, all went well. Unfortunately, blogs are more interesting when things don't go so well :-)

I gotta say, though, the sweetest part of the lab was actually looking at our chlamy cells under the microscope. Karl insisted that we do so, and I'm totally glad I did. It's really neat seeing tiny 'critters' swimming around with their little flagella. I then looked at my finger in the microscope. I gotta admit, it wasn't as interesting.