Designing better RT-PCR primers

Last post I talked about amplifying DNA by real time-PCR, which measures the number of amplified copies at the end of each cycle, giving researchers "real time" numbers of DNA copies. To do this however, you need appropriate primers to get the job done. Not only do they have to be specific enough to work only for the DNA you want amplified, but I'm learning there are other tricks you need to abide by.

Let's back up a little bit though and look at transcription and translation. After all, I'm after the mRNA transcripts that are made in this process. With my genetically engineered construct, protein synthesis starts when transcription factors bind within the cloned 5' untranslated region (UTR) and begin transcription at the promoter, transcribing all of the way through the eGFP open reading frame (ORF) and through the 3' UTR. Now we have an mRNA transcript with part of the 5' and 3' UTR intact at either end of the eGFP coding region. This will serve as the template for translation, which begins at the start codon of the ORF and ends at the stop codon. The 5' and 3' UTR are not translated, hence their UTR moniker.

My project is aiming to measure the amount of mRNA transcript in cells under different environmental conditions. It's not easy to measure mRNA by itself, but it is very easy to measure DNA. Using mRNA as a template, you can make complimentary DNA using the enzyme reverse transcriptase.

Reverse transcriptase starts at the 3' end on an mRNA molecule and transcribes a complimentary strand backwards along the mRNA. However, reverse transcriptase will eventually fall off (represented by the fading orange triangle), so smaller mRNA transcripts work the best.

If I have this mRNA transcript that I want to measure through RT-PCR (after I've converted it into cDNA with reverse transcriptase), I need a primer to amplify the eGFP coding region.

I first made this primer pair to test for the presence of eGFP in my diatom cell lines. It amplifies most of the eGFP coding region by attaching at points just inside of the gene, as you can see below.

Unfortunately, these primers aren't working very well when it comes to applying them to real time-PCR.

My control real time-PCR reactions have worked pretty well for me each and every trial I've run. The control reactions use primers to amplify the endogenous genes we're manipulating in our system, which serve as a good comparison to the experimental reactions.

You can see on the graph at right that the primers amplifying endogenous genes work pretty well, developing curves within an appropriate cycle range (the number of cycles until a noticeable amount of product can be measured).

However, when I look at the graph for the transgenic lines using the above primers to amplify eGFP, I get a graph like this on the left. The amplification lines are severely delayed and do not approach the same level of product by the end of the reaction.

In addition to amplification plots, the real-time PCR application on the testing computer also shows graphs that display the melting point of the double stranded DNA molecules. These graphs can be very informative when troubleshooting real time-PCR reactions.

Here is the dissociation curve of the endogenous amplifications:

 Oh boy, it's that curve crisp and clean.

Here is the dissociation curve of the transgenic amplifications:

Yeah, not so much. This curve is extremely messy and non uniform. This provides further evidence that my primers for this reaction might not being working as well as they should be.

After talking to my adviser and seeking some advice online, I've found a couple of parameters to follow to make better primers.

First and foremost (and going back to my bit about reverse transcriptase starting at the 3' end of the mRNA transcripts), my adviser let me in on a secret: I should be using primers that amplify near the 3' end of the transcript since that will be the highest quality region of cDNA as it is transcribed. Using this knowledge, I am working on primers that amplify in the area represented by the orange square, just outside of the 3' UTR. I also tweaked the settings which the primers conform to, based on information I found on other university websites. Yay for Google and other scientists!

The past few days in lab I've been working on these primers and planning out my semester of science ahead of me. Just this week I put together my thesis committee (Justin Thackeray who I had for genetics three years ago, and David Hibbett, my undergraduate adviser) and I've begun re-reading some primary literature and will soon begin reading more broadly and in depth in preparation of writing my thesis. I've also started putting together bits of my paper, which I should have done a while ago.

Anyway, that's all for now, really.

If you've gotten this far, watch my latest YouTubes video regarding this topic matter:

And more snow: we gots it

We got some more snow here on campus, and yet again I didn't know about it until just before it happened! I think we got another 5 inches of snow or so, which brings our total to over 2 feet since I left campus at the end of the Fall semester. By noon, it appears the snow has stopped and the sun is thinking about coming out to hang out with us. This would be appreciated because we all like the sun. It will be interesting to see what my fellow classmates do tonight for their first weekend back at Clark. I wouldn't be surprised if a lot of students stay in to avoid trudging through the snow late at night, but who knows? Apparently LOST is on Netflix instant, so I know what I'm doing tonight.

I originally had an interview for a research assistant position this morning, but my professor couldn't make it in because of the snow. It's rescheduled for next Friday. I don't know what my chances are for landing the job, but if I do, it would make my life a lot more complicated during the week. Either way, my schedule's organization is up in the air for at least another week. I've been trying to juggle classes, labs, and readings the past few days and it's a bit of a hassle.

I finally stopped by the lab today to organize some stuff. I don't know how long it will be until I can start with the actual lab work, but at least I'm starting to get on track with some things. I hope we can start things up next week and it'll be awesome!

 I posted a new video to wrap things up for this week. Below it is:

It's Getting Colder Out... And Exams Are Heating Up? or something like that >_>

It's early December and we're without a significant snowfall. It's cold and campus is winding down, while the library and the Academic Commons are getting more full every night with students studying and meeting up for group projects. I'll admit that with finals and without any snow and the trees without leaves, campus is a little dreary.

But campus is always prettier (if not much colder) when it snows. Even though we can't appreciate it much because we're busy wrapping up the end of the semester, it's always nice when the first major snow hits. It's been a little longer than usual for me since I last saw snow because I went to Australia, but I feel like it'll be normal for me to crunch through the snow once it falls again.

My work in lab is still ongoing. I'm working on collecting my "control" plasmid for the expression experiments, and I'll be prepping everything for the transformation process. Hopefully I'll be able to start those experiments at the beginning of the spring semester if I can transform the diatoms before winter break.

In the midst of trying to get everything wrapped up at the end of the semester, which has me a little more anxious than normal already, I'm all of the sudden in the middle of signing a lease on an off campus apartment for next year. Just a block and a half from where I used to live my first year on the edge of campus, I might be living on the first floor of an apartment filled with people I know and like. I'm really nervous though, because I've never signed a lease before and things are not finalized with 5th year.

Anyway, I'll be posting a few posts here and there, some related to lab and others not, so be sure to check back!