I get a variety of feedback from this blog; some fellow PhD-ers enjoy being able to relate to my emotional hiccups, whereas others get annoyed that I haven’t actually told you what I’m doing, just merely indicate the way it makes me feel.
So this post is reciprocation to a combination of this feedback.
Some of you may have noticed that it’s been a while since I last posted. This is because I entered an unfortunate phase of PhD-ing where you forget the rest of the world exists,
At this stage of my project, I am attempting to analyse gene expression of two variants of one gene, across multiple tumour and cell samples. We do this by performing RT-PCR. RT-PCR reactions require just three things: cDNA template (painstakingly extracted from your sample of interest), primer/probe (corresponds to your gene of interest) and an enzyme.
You can use probe OR primer in order to detect your gene expression. Probes are much easier because they are pre-made by companies and come with specific instructions to get them to work. However, sometimes you have to revert to primers (which you have to design yourself) if the probes for your gene i) don’t exist or ii) don’t work. Primers are way more tricky than probes as you have to work out the right temperatures and concentrations to use for yourself. This process is notoriously painful among researchers.
In my case, I started off with probes but I couldn’t get the QC (quality control) step to work. Although the companies who sell the probes guarantee that they work, you have to do this step, just to make sure your data are real. This QC step consists of performing the experiment on a 1 in 2 serial dilution of cDNA samples. The theory goes that, if each dilution has a concentration of half (start with 100%, dilute to 50%, then dilute to 25% etc) of that than it’s predecessor, then there is half the quantity of your favourite gene. When you run your RT-PCRs, this should be evident; if it’s not, then your probes are not working at 100% efficiency. I performed this step on a number of different probes and they all worked really well. However, for two of them, the efficiency was really poor. The differences in cDNA concentration were never detectable, AND even though we perform the reactions in triplicate, the repeats never looked the same as each other. There was obviously something weird going off with these particular probes. Reluctantly, I set about designing and ordering my primers.
Another note about RT-PCR reactions. We always run a NTC (no template control), to make sure that none of the reagents are contaminated with DNA. We want to be sure were are measuring the cDNA of our sample of interest, not some random contaminant. As soon as I ran the experiments with the new primers, I saw that there was contamination of the NTC. Weirdly however, it only happened sporadically. At first I just assumed that I had accidentally put cDNA in the NTC. It actually took several repeats for me to reassure me that that was not the case.
After going over the data with my supervisor, we agreed that the most likely cause of contamination was the (expensive) enzyme. It had been opened since April last year, so it wasn’t unlikely someone had mistreated it since then.
However, even after ordering new enzyme, we were still getting the same problem.
There were a few more suspects however. Although I use a new tube of water for each reaction, these are all taken from the same source. It could be that this source was contaminated.
Sure enough, after using a new water source, the problem was fixed. On a reaction plate with 2 NTCs, neither of them were contaminated.
I set about performing more QC steps. However, in my first batch of data, the problem returned! Had I messed up? Had something else been contaminated since the last time? Or had we just been lucky to avoid NTC contamination before…? It had only been happening sporadically in the first place.
Another suspect was the pipettes. After dismantling several and peering inside, it looked pretty likely that they were the cause, as they were pretty gross. HOWEVER, I had been using filtered pipette tips to stop this from happening. It seemed pretty unlikely that the pipettes were to blame…but we gave them a clean anyway (well, the lab manager did anyway. Thanks Trish!)
At this point, when my data was starting to defy science itself, my supervisor was kind enough (THANKYOU!) to step in and give me a hand. I watched as she set up the same reactions in the same way that I had been doing for the past few weeks. The theory was that maybe I was doing something (?) weird in my set up to cause the weird results. It quickly became apparent this was not the case. We ran a HEAP of NTCs to make sure we didn’t miss anything, this time. I was actually pretty thankful when we got the results back and the NTCs were sporadically contaminated. It could have just been that I was cursed!
We came to the conclusion that the probes themselves were contaminated. This was not good news, as I had re suspended them (they arrive as a dry powder and you have to dilute them) with a chemical that some others in the lab had used,so it was likely that they would have to repeat some of their experiments. Selfishly though, I was pretty relieved that the mystery had been solved.
Now I just had to order some new primers, but it would be a few days before they came in.
In the meantime, I thought it would be useful to try and get our money back from those dodgy probes. I knew that arguing with biotech companies could be a pain in the arse, so I set up a reaction with so many different controls that there was no way that they could blame US for THEIR products not working. Now, you may remember that all the other probes, bar two, had worked fine. I had used the same source of cDNA to run the QCs. However, I thought it would be good to run some other cDNA sources, so the biotech couldn’t argue the cDNA was dodgy.
After getting the results, I noticed that the original cDNA source results all failed, as predicted. None of the replicates looked the same. Typical. However, as I was sitting down to email the results to the company, I noticed that not all the replicates were behaving as weirdly as the others. Why? I thought. Is there any patten here? Yup. You guessed it. All the reactions with the new cDNA source behaved really well. Maybe it was a fluke, I thought. I know allll about lab flukes. By this point, it was 7PM and I knew going back into the lab would not be a good idea (when I’m tired and grouchy, I tend to make mistakes). So I went home, planning on running a few more QC checks, using the new cDNA, in the morning.
I was at work by 8AM that day, genuinely excited for what I might find. I set up the reaction and sat back to wait for the results. An hour or so later, I watched the computer screen as graphs of my data revealed themselves. Two perfect rainbows of 100% efficient probes with perfect replication revealed themselves to me. The probes worked just fine.
I wasn’t quite sure how to react. Imagine if I had picked a different cDNA sample three months ago. I never would have gone through the stress of the primers. I probably would have a significant pile of data by now.
However, this isn’t a very great mindset to hold. If I had picked a different cDNA sample, I wouldn’t have learned or progressed intellectually/ personally anywhere near as much as I have. Namely:
-Trust your own data! When something fails, don’t immediately assume it was your fault. (part of the reason all this took so long was that I had to keep repeating experiments as I just blamed myself for their failure)
-I now know how to run DNA products on a gel (this is useful for estimating fragment size, and making sure your experiment worked)
-I now know how to use a Bioanalyzer (a fancy machine which you can use to find out about that tiny quantities of RNA/DNA in your sample)
-I now know how to optimise primers!
-After using so much cDNA to get the experiments to work, I’ve got pretty good at RNA extractions.
-I’m so much more confident in my own abilities now! (i.e. I’m not actually cursed.)
-I’ve bonded with lots of my colleagues as they have all helped me with my experiments,
-I’m able to help OTHER people in the lab with my new knowledge, for the first time 🙂
This is a long blog post I know. I’m not going to apologise though :p
Oh yeah, some of you might be interested to know why that particular cDNA source made the probes behave so weirdly. Hmmm, I’m still unsure on that myself. It may be that the gene had a mutation? If you have any ideas, please let me know…
NOTE: Sometimes this blog is difficult to write. In my head I see PhD students, prospective employers, my parents and my supervisors reading it. So it’s hard to pick a tone. Apologies if I have gone over or under your head here :S