I've received my new primers and already run a couple different reactions with some results that prove to be promising and others that are frustrating.
Yeah, encouraging :]
The first thing to do with a new set of primers is to run them in a normal PCR reaction to see if the primers amplify the correct length of DNA. While I could do this with my super concentrated, ultra clean plasmid DNA that I used to transform my diatoms (which would serve as the ultimate positive control), I decided to be be bold and try to amplify my complimentary DNA (cDNA) samples. This cDNA was made from mRNA that was extracted from diatom cells exposed to different environmental conditions. Because the mRNA we're looking for contains GFP, anything that is amplified essentially means that our plasmid DNA that we transformed into this line of diatoms is being expressed, which is a great, great thing.
So I ran this PCR reaction with my new primers to amplify my cDNA and this is the gel I got:
There are some pretty convincing bands in that gel which is really encouraging. It appears that all but two of my reactions (6 out of 8) gave us at least some PCR product.
Nicccccce :D
I then ran a real-time PCR reaction called a standard curve, where the source DNA is serially diluted ten fold (I ran 1:1 through 1:10,000 dilutions). For this reaction, I do go ahead and use the plasmid DNA control to give us the cleanest results possible. This reaction allows us to see how efficient the primers are at doubling the amount of DNA product at each PCR cycle.
You can see in this gel (in the first 5 lanes) that a lot of PCR product is being produced. Each reaction hit their saturation point which is why each reaction looks the same even though they had drastically different amounts of starting DNA template. The great this about real-time PCR is that we can see on the computer screen how each reaction was amplified in real time, and see where each reaction it a ceiling amount of DNA. In this reaction I could see that even the 1:10,000 dilution easily hit this ceiling before the 40th (and final) cycle.
With these two encouraging results--the proper amplification using my new primers in a standard PCR reaction and strong amplification in my standard curve using plasmid DNA--I went ahead and tried amplifying my cDNA in a real-time PCR reaction.
Unfortunately, I ran into the same problem I've been having for a few weeks now (see the bottom half of this post).
Sad face real-time PCR :[
Not all of my reactions amplified, and those that did didn't amplify as cleanly as I wanted them to. I ran a gel of my second real-time PCR reaction, which visualizes the two amplified reactions (lanes 3 and 6 starting from the top).
While this is certainly a set back, I have a couple of things I'm going to try. Most importantly, I've ordered a new kit to run the real-time PCR reactions, since the kit I was using was "old." Next, there are a few things I can do to ensure my reactions are as balanced and clean as possible. Finally, I'm going to drop the annealing temperature of my real-time PCR. While I was using the same annealing temperature from my standard PCR, there are two main things that differ between my standard and real-time PCR reactions. First, the salt concentrations are most likely different (although that's a bit annoying to look up, but it's on my to do list), which I found out while screening my diatoms can really screw up a PCR reaction. Second, I used the mysterious "Q-solution" provided by the company Qiagen in their PCR kit when setting up my standard PCR reactions. This may also have significantly changed my standard PCR reaction. While the real-time PCR reaction really should be working with my current set up, it's very possible that my primers are finding it difficult to "seek out" and bind to the sparse cDNA that I want them to amplify.
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:
