Extracting RNA from DIATOMZ

In order to measure transcript abundance to collect data for my project, I need to extract RNA from frozen diatom cells. These diatom cells remain "frozen in time" from my experimental assays, which expose lines of diatoms to ammonium. By freezing the cells in liquid nitrogen, I can later extract their RNA and get a good idea of what the transcript abundance was at that time.

To find out more about my experimental assays click on the link up there (which is also here) and then watch my video down below about RNA extractions. These videos together describe some of the most important work for my project!

Diatom Culling, Revisited

A few weeks ago, I posted about staving off a massive culling of diatoms.

As of right now, my diatoms are still on the mend but I hope to have things all sorted out and lined up by the end of the week. To do this, I'll be checking on my plates and ensuring all of my diatom lines are accounted for before I clean out the culture room, which is in desperate need of being tidied up.

In the video below, I give my account of what it was like to prevent my diatoms from dying. Hopefully I will have been successful in doing so.

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:

Oh, winter is here, okay, thought it wasn't coming

Ever since Worcester got slammed with snow and ice just before Halloween, 2011 was pretty mild. In fact, the month of November felt warmer than October, and by the time I went home at the end of the semester, we didn't have any snow in the 01610.

But about a week into the New Year, we finally got a few inches of snow.

A view of the JC (left) and the AC (right) from Atwood Hall.

And over the past few days, we got a couple inches of snow and ice in small increments.

A view of the AC on Sunday morning.

This snow follows a string of extremely cold days in Worcester, with temperatures in the single digits and teens.

So when I thought winter wasn't coming around this season, I was definitely wrong.

The Zen Garden looks like a mini snow bowl.

Campus is now covered in snow, but we're still well behind the amount of snow we got last year.

But I'm okay with that. Cars have already been struggling to make it up the lame excuse of a hill my road is on due to the snow and ice. I'm pretty sure one of these days a car is going to careen into another, because everyone seems to think it's a great idea to simply floor it when they lack traction on the ice.

While we may get a few warm days in this coming week, we may get two doses of freezing ran. That's something I really don't look forward to.

(Tuesday the 24th update: it rained last night and was in the upper 40's today. All of the snow not piled up is gone. Ha! New England weather!)

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.

Unflagging speed

The start of the semester is beginning with unflagging speed, and I find myself dazed from the extreme acceleration. I’m desperately trying to prevent further acceleration and maintain a steady pace so I can catch up and organize my plan of attack on these beastly final few months.

I have tons to be worried about this semester too, which is just fantastic:

• I should hear back from grad schools within the next few weeks
• I need to correct and tweak my lab experiments as soon as possible so I can collect data worthy of a Master’s thesis
• I need to then find time to conduct those experiments and collect that data
• Following that, I need to write my thesis and prepare a lengthy presentation
• I should probably also defend my thesis in front of a treacherous jury (complete with torches and spears)
• Aside from my Master’s work, I’m going to be helping out with a research seminar in my lab, which has the potential to eat up a lot of my time, especially in the second semester
• And it would also be great if I could make some money

Slaving away at the bookstore.

All of this to me seems incredibly daunting, especially since I haven’t had much spare time after I got back from my trip to Florida thanks to a lot of time at the bookstore. But there is a glimmer of hope at the end of this dark, damp tunnel that is the beginning of the semester.

Things are the bookstore have looked like this recently: were does the work start and where does it end?

I hope to essentially eliminate all of my commitment to the bookstore after next week, freeing up a lot of my time, which I can devote to lab work. I also hope to get my project on track really soon, and have a set of goals by the end of the month. If I can get my project on track by then, I can grind out a lot of lab work over the month on February (thankfully we have that extra leap year day).

Then I could have the month of March (which includes a week of spring break) to collect my thoughts and write my paper and put together a presentation.

I can’t decide whether I’m tricking myself into thinking this is feasible, but I guess that’s the only option I have right now. The only option I have is to tear this semester apart and make it mine.

The remainder of this week is already completely full of plans to start my project back up again and working at the bookstore. While I’ll miss the extra income from working at the bookstore, I can’t wait to devote all of my spare time into my project so I can get comfortable with it’s progress and outcome once again. Once things slow down with the beginning of the semester at the end of next week, I’ll be taken off the schedule and I can devote entire days to lab and do a lot more with my blog and do more blog projects, which is something else I’m looking forward to. More science with my next post, which I'll post soon!

Staving off a massive diatom culling

At the end of last semester, chaos tore through my portion of the laboratory when a massive die-off of diatom cultures was a major possibility.

My diatom cultures have been growing in glass test tubes with liquid media, within which they're happy for an upwards of three weeks before they need to be transferred to fresh media. There are a number of factors which make culturing my individual lines of diatoms different from our various stock cultures of diatoms that also grow in the culture room, so it's been a bit of a change for me.

With a few weeks left in the semester, I inoculated two large cultures for my final experimental assay. After about five days, there seemed to be almost no growth, but I figured I'd give them a few more days. After a week I told myself I must have messed up somehow, so I inoculated two more large cultures and I was on my way. At about this time, I made some solid media plates and spread all 40+ of my cultures on individual plates to preserve them while I was away on holiday. (The solid media plates combine the sea water/nutritional supplements I usually grow the diatoms in with agar to solidify the mixture. This is essentially the diatom version of the plates I used to grow my bacteria on when I was cloning DNA.)

But then the second round of large cultures didn't growth either. (Meanwhile my plates still had another 5+ days to show any signs of growth.)

...Yeah... It was at this point I went into a deep panic because my diatoms in my test tubes were dying off and it came to my attention that the materials I was using to supplement my media (i.e. the nutrients to sustain growth) were, well, bad. (This is what happens when I'm not responsible for all of my materials. I am not attacking anyone, but when things matter in the lab I should really do everything myself like make the materials needed for my cultures.) This meant the plates I had made using these materials would not sustain any growth and all of those plates would have to be thrown away.

Now I was upset for multiple reasons: I lost a full day's worth of tedious lab work setting up my first 40 plates and all of my diatom lines were thinking about kicking the bucket.

GREAT.

Additionally, I didn't get in my final experimental assay because my diatoms kept dying in the large cultures due to poor nutritional content.


The end of the semester resulted in frantically making new plates with different materials and pleading with the science gods that they grew while I was away at home.

(Insert a lot of World of Warcraft playing at home)

After a week in the hills of Vermont and New Hampshire, I checked back into lab before heading off to Florida. Thankfully, almost all of my plates showed signs of life which meant I could catch my plane without fretting about my meager diatom cultures.

Attempting to save all of my discrete diatom lines.

After I got back from Florida a week later (roughly two weeks after the second plating), it was time to check on my plates once again.

This time I wasn't as happy with what I saw. Because I was hasty in finishing my plating before I left for home, I did sort of a good-enough-but-crude job. While some of my plates may still be good and support/sustain healthy cultures after they dry off some extra moisture from the initial plating (I sealed each plate to prevent them from drying out), I decided to transfer each culture back to new test tube cultures. I ended up using a combination of plated cultures (two weeks old), old liquid cultures (one month old), and really old liquid cultures (two months old), to sustain almost every line I started with. There are still a few lines I may be able to salvage from my plates
 

A majority of my massive diatom culture collection.

This has been an interesting experience for me and I still have a few things to learn and figure out. The tricky part is that I don't have a lot of spare lab time to tinker around and perfect everything. But because I like everything to be done right, I'll figure them out. First and foremost, I'd like to plate my diatoms so they can subsist for at least three months without me tending to them. While it will be a fair amount of work invested at the beginning of the process, it will save me heaps of time in the long run because I won't have to cater to the high-maintenance demands of my diatoms.

Once I settle back into my hectic life. I hope to consolidate all of my diatom cultures for long-time storage and press onwards to finishing my project for once and for all.