PCR in Real Time

I really like graphics, and I make a lot of them to represent my lab work.

Below is a graphic that represents my lab project. So far, I've gone through the greens, yellows, pinks & blues and am working in the orange to red zone. Well, I still ave some blue work to do, but I'm currently working on orange zone material

My project in a very small nutshell the size of your computer screen.

That is, I'm trying to amplify cDNA derived from frozen cell samples to make estimates of gene expression from my different lines of transformed diatoms.

 "Regular" or endpoint PCR can hit a duplication plateau that isn't limited by the amount of starting template in a reaction, and therefore usually only gives a positive or negative result (yes there is product or no there isn't). Real time-PCR on the other hand lets us see the amount of amplified product as PCR cycles progress and lets us accurately quantify the amount of DNA that is being amplified each round. By using specific primers that only amplify one region within a pool of DNA, we can determine how much particular DNA we had to start with.

Let's say the cartoon to the left here represents our real time-PCR experiment, and we have a pool of cDNA waiting to be amplified. We have primers ready to amplify the pink cDNA and the dark blue cDNA. Using these primers, we set up a reaction to amplify the pink cDNA and a separate reaction to amplify the dark blue cDNA. If we incorporate a special dye into each reaction, any double stranded DNA that is amplified by the primers with fluoresce, which can then be measure by the real time-PCR machine. This data is then put into graph form, and we can track the amplification of DNA in real time as the experiment progresses. In the cartoon, you can see the pink cDNA starts at 2x and the dark blue cDNA starts at 1x in terms of starting cDNA template. As such, the amount of cDNA that is made (and therefore the amount fluorescence given off) will vary.

In the graph on the right, we can see the difference in fluorescence between the different amplification reactions. Here the red, blue, and gray lines are almost identical, and are far ahead of the green line. This means that these three lines have much more starting cDNA template than the green line.

This is the approach I will take in measuring the gene expression in my experiments, if I can get my primers to work. But more on that in a later post.

Thursday Science

Colorful labels mark primers for my real time-PCR reactions.

Setting up said real time-PCR reactions next to the special PCR machine that will quantify my cDNA of interest.

The computer screen displaying the real time-PCR software we use.

Going to do a bunch more RT-PCR tomorrow. Wish me luck :-\

From GRE to RT-PCR

The view of Lasry the other day on my way into lab.

A few weeks ago I took the GRE as part of my quest to continue graduate school in a Ph.D. program. Standardized testing is such a money scam given all of the fees involved from taking the exam to getting your scores and sending them to your schools. Furthermore, I don't think standardized exams are particularly informative, which is funny because Clark was just in the news because they are no longer requiring SAT scores for undergraduate admissions.

But I digress: I'm not here to complain how ridiculous the whole process of taking the GRE is. I'm just super glad the GRE is behind me and I can focus on things that actually matter. Yet ever since, I've been taking it slow with my workload because I needed a bit of a break. Thanksgiving marked the end of that break though, and now it's time to jump right back into things to finish up a productive semester.

I have a very hopeful list of things I'd like to accomplish by the end of the semester in mid December. But because I'll need to be working a lot more hours at the bookstore to help with the end of semester rush, it's going to be a big squeeze.

We're starting to get books for the spring semester at bookstore, & they're piling up.

This  picture is enhanced with the iPhone app Cat Effects.

Meanwhile in the lab, I have heaps to do. I want to finish up my experimental assays, screen and grow out more diatom cultures for future experiments, prepare a presentation and do some writing, and finally start with some RT-PCR experiments.

Now previously I extracted RNA from my frozen cell samples and used the mRNA present from the cell samples to generate cDNA.

RT-PCR, or real time PCR, will allow me to quantify the cDNA that I have made from my RNA. This is because RT-PCR can measure the number of DNA copies at each copy cycle by measuring the fluorescence of a special dye that hybridizes with the DNA. This is pretty snazzy, but it looks like I'm getting myself into a lot of grunt work.

You see, in order to get precise data from RT-PCR, one usually runs three replicates from one DNA sample and compares the output. But it's not like I only have one DNA sample. I have tons.

This is because I have 8 DNA samples at the minimum per experimental assay that I run in the beginning: I have two different cell lines that I test per assay, which get divided between two different test conditions, from which I take cell samples at multiple timepoints.

For the time being, I'm going to start with time zero and my end time of 60 minutes. This will hopefully give us start and end data that will display the overall trend of my experiments.

But because of positional effects, I need to run more than one experimental assay. I'll probably run three sets of samples through RT-PCR, but I hope to have up to 5 samples completed and ready for RT-PCR by the end of next week.

This is from today while I waited for my next sampling time.

Wait, what's that positional effects you just mentioned a second ago? Oh, yeah, positional effect. Because we transformed our diatoms with ballistics, our DNA was randomly inserted into the diatom's genome. By chance, the DNA we're trying to measure in our experiments might be inserted into a region that is either expressed more or less often, which would skew our data. By gathering data from multiple replicates, we'll get more accurate data regarding gene expression. Cool how science works, huh? We think of the neatest little caveats. Well, I mean, my professor does.

But sometimes I do too!

Like last month or so I came up with a solution as to why our cultures weren't growing so well. And then more recently I figured out how to screen my diatoms for our reporter gene more effectively.

So yes, sometimes I come up with cool things too.

The Jonas Clark building. I can't stop taking pictures from this vantage point! Gah! I love it! 11/18/2011