RNA extraction and quality control of your Volvo

I got an e-mail the other day. It was from Agilent who said that my order of two custom microarray slides scheduled to arrive around the 5th of September were already finished and were being shipped from Germany to the analysis facility BEA at Karolinska Institutet (KI) now.

Guys! You're two weeks early! I haven't even gotten my own stuff (samples) together yet. Germans... How often does this happen? By experience - always when you're lagging behind. That's not what I replied though, but it's what I silently told myself.

Now you might think 'hey, you only have to fill one slide, 8 samples, how hard can it be?'. If you just thought exactly that, I'll give it to you. You're right, it's only 8 samples and it would take me 2 hours to extract. However (I hate that word, and I use it all the time), out of the 12 samples that were already extracted only 2 had enough total RNA for analysis, and don't get me started on the quality control of that RNA... ops, I already did that myself... they all failed.

At this point I have nothing. Fortunately for me, we have lots of archived RNA with corresponding DNA samples which have already been processed in the qPCR. Basically, that means that I can see in which RNA sample that I should have the right organisms and enough material. However (here we go again), that doesn't guarantee good enough quality RNA.

There are several reasons why the RNA in my samples might not be of good enough quality, and when I'm talking about quality it's rather specific and doesn't entail the comparisons between e.g. longevity of brands that we're so used to in modern consumption society. No, here, I'm talking about degradation and size of the RNA fragments. Like if you removed a few parts from your Volvo, it would still be a Volvo, but it would probably not function very well, and if the parts removed were body parts, the untrained eye might not even recognise the car as a Volvo anymore.
A similar problem arise when I have low quality (degraded) RNA. The probes in my microarray are all extremely specific (untrained eyes) and when the genes (Volvos) in my samples are missing a few parts due to degradation they will not be recognised.

Image credit: Fabian Oefner

To be short and simple, RNA is the coded message that our DNA relays for our cells to, down the line, translate and produce proteins with various tasks and properties. Naturally, the message doesn't stay intact for very long since, in most cases, when the message has been delivered and translated it has outlived its usefulness and is recycled within the cell.
So, I might just have been unlucky and the RNA in my samples was naturally degraded when the research vessel arrived (too late) on site. I might do a terrible job extracting the RNA, and perhaps, mysteriously introduce RNase (an RNA degrading enzyme) to my samples. The agitation step (shaking the samples really, I mean REALLY, fast together with tiny glass beads to crack the cells open) might shear the RNA to tiny bits and pieces. The latter is the only explanation that I can actually test.

I'm assessing the RNA quality in an instrument called Bioanalyzer 2100 (Agilent), which gives me a value from 1 to 10 (10 being the best - most intact RNA). It has developed into an industry standard since it uses an algorithm to objectively calculate and compare the amount of two different ribosomal RNAs. This way us scientists know that we will get good and reliable results and we secure the possibility of reproducing the experiment (important for e.g. peer-review and validation). Everyone is happy. Me included, if I can get good quality RNA, otherwise no.

Grit and the growth mindset: The recipe for success

I recently watched a TED talk by Angela Lee Duckworth. It was recorded in 2013, so I have no idea how this inspiring talk has eluded me for so long.
Anyway, Lee Duckworth tells her tale of how she left a demanding but successful job in consulting to teach math to 7 graders. That's where her ideas and research started to develop when she observed the struggles of her pupils. After a few years she started studying psychology and pursue the unverified observations she made as a math teacher.

You can see the entire 6 min talk right here.

In her talk she also mentions, at the very end, a concept called the 'growth mindset'. This is also an idea that has been around for quite a few years. It was coined almost 30 years ago by Carol Dweck who studied how students dealt with failure. The opposite of the 'growth mindset' is the 'fixed mindset' and what sets them apart is the belief that talent is either static or developmental. This means that if you believe in the 'growth mindset', talents, abilities and even intelligence is not something that is restricted to life's lottery, but is actually something you can improve and develop.

I first heard about the 'growth mindset' during the workshop I co-hosted in Communication, feedback and presentation techniques, where I had a very interesting discussion with my department's Public relations manager. I didn't think much more of it after that, to be honest, but when I now heard it in the context of Lee Duckworth's TED talk, it made perfect sense. It is also how I would define my own success.

So together with the 'take home word of the talk': grit - the 'growth mindset' is what defines success. As nicely put by Lee Duckworth "grit is living life like it's a marathon, not a sprint". It's about setting up long term goals and sticking to them, not by hours, days or weeks, but years. Perseverance.
It's about not giving up, and use the inevitable failures to grow, work harder/smarter, and eventually succeed. Because the thing about success is that it doesn't always happen right when we want it to happen.

Lee Duckworth wraps up her talk with the task that stands before us all, and that is teaching our kids to be grittier. Because talent and intelligence means nothing if you're not willing to work hard.
When I was a kid I played hockey and I strongly remember a small piece of paper that hung on the whiteboard in the club's meeting room. It said: "The will to win must be stronger than the fear of losing". I didn't quite understand the deeper meaning of it back then, and I'm not sure whoever put it up did either.

In hindsight I wish that someone would have explained it to me. I wish that I had even a fraction of the knowledge I have now, back then, and I'm surely not alone with that wish.
I think Lee Duckworth is right, and when I think more about it, I'm actually subtly teaching my kids grit. Perhaps it's time that we do the same, although more formally, in our schools.
No matter how small the task I always tell my kids to never give up and keep trying until they make it. Often to their utter frustration, when they can't get their seatbelts fastened in the car. However, not a single occasion have they not made it, eventually, and their howling annoyance been instantly replaced by silence. Dad was right.

Cross-hybridization

This is going to be a tough one, and I'm afraid I might end up with more questions than answers at the end of this blogpost. Either way I feel like I need to clear my mind on the matter of cross-hybridization.

After a long and nice summer holiday with my family, work didn't progress much, even though I had hoped that my external contacts would be able to do something after final submission of my microarray design. However, it turned out that everyone else was gone on their summer holidays too. I should have known.
Now everything is moving along nicely again, but the road up to the final design was certainly not a straight one, mainly due to cross-hybridization.

This issue has been a constant grain of sand in my eye since the day I set foot in a molecular lab, however, it got all the more profound when I started working with qPCR and gene expressions.
As of now, there's no satisfactory way of dealing with it, we just know that it's there and can do our best to account for it in our data analyses and interpretations.
My next project (after the microarray) will attempt to at least improve (decrease cross-hybridization) for some of the known and widely used oligonucleotides in marine cyanobacterial diazotroph research.

Cross-hybridization is basically hybridization of a primer or probe sequence with a sample sequence other than the intended target. Often it happens due to several sequences being highly similar, but also because the oligonucleotides used are not specific enough in their design.
What I mean with specificity in this case is that even though your oligonucleotide design is a perfect match to a certain region of a gene in a target organism, it could match almost, if not just as well, for a closely related species with the same gene. Moreover, even a close match (not perfect) could be enough for cross-hybridization to some degree. There are several reasons why mismatches in nucleotides still hybridize, like for example the nucleotide content of the target sequence (G-C-T-A) and the location of the mismatch in relation to the 5' and 3' ends.
To make matters even more complicated, the concept of species is kind of blurred when working with prokaryotes, where we often are interested in different strains of the same species. Even though they're systematically the same species, phylogenetically they're not, and potentially have different impacts on different ecosystems.

So running cross-hybridization tests where I use oligonucleotides designed for a particular target organism on a closely related strain or species to see if I get a signal. If I do get a signal, it is a cause for concern, but if I don't get a signal it is safe to say that the designed oligonucleotides are able to distinguish the target organism from at least that particular closely related strain or species which I tested against.
Of course it is impossible to test for every single possible cross-hybridization. There are simply too many unknowns involved and for some strains we don't even have any designed oligonucleotides yet. It is also possible that there are closely related strains or species out there in the ocean that we just don't even know exist.

An emerging number of marine cyanobacterial diazotrophs are just known by sequence, matched by phylogeny, in metatranscriptomic studies. My diatom diazotroph associations (DDAs) for example, are easily observable in a fluorescence microscope, so I can quickly look and see if they're there and which species and strain it is. That will initially be key to improving and optimizing oligonucleotide designs for the larger marine cyanobacterial diazotrophs. For the smaller we're somewhat dealing with a black box and I'm expecting a trial and error approach.
Up to this point we've mainly focused on the nifH gene for our target sequences, but as these organisms get fully sequenced, we might find alternative genes which are better suited for our purposes, based on relatively subtle genetic differences.

Seminars and presentation techniques

I thought about writing a blogpost about cross-hybridization after my last post which barely touched upon the tricky issue. However, two rather technical blog posts in a row might be a bit too much to digest.

So, instead, after having two scientific seminars (about my research) in three weeks, I decided that it would probably be a good idea to write about my experience and thoughts on my presentations and also presentations in general.

I feel that I've talked enough about my licentiate seminar, so I will kick off by mentioning the nice introduction I got from my PhD colleague at the beginning of my last seminar. I was fairly well informed about the content of the introduction (since he asked me about it), but one thing that still got me thinking was when he mentioned that I co-hosted a PhD workshop/course on communication, feedback and presentation techniques earlier this year. It was very successful (based on participant evaluations) which opened up for further development and a chance to host it again next year.
One of the most common questions that I got during the workshop concerned a list of recommendations, or do's and don't's, of oral presentations. I was a bit reluctant to answer this question, especially since we touched relatively little on this subject in our theory and the presentation techniques were mostly based on self-development and feedback from the participants. I also believe that there is not one single recipe that fits everyone in this case, rather I think each and every speaker have to find their own personal style and the tricks that make them comfortable standing in front of a crowd.

So, where am I getting with this? Well, after my two recent seminars, and getting lots of positive feedback from both fellow PhD students and faculty, I might not be giving myself enough credit in terms of my knowledge in presentation techniques (and perhaps even my research).
I think anyone who has ever acquired a Masters degree (or similar achievements) is familiar with the question: "what the have I really learned during these years" when looking to, for example, apply for your first job.
Sure, I have no formal training in oral presentations, although I do have a neat little hardcover book in my office on how to improve your oral presentation skills, but I haven't even nearly finished reading it. But practice makes perfect, right? And with enough experience you're sure to at least know a few tricks of your own. However, never underestimate the importance of watching and learning from other great speakers. They are great because they had the courage to walk that uncomfortable path of trial, error, feedback and improvement.

With that said I would like to share my experience, feelings and thoughts on the recent seminar and oral presentations in general. Perhaps some of it might be incorporated into the next workshop. Who knows.


1. Preparations

A good presentation is based on good preparations. Of course this entails having a solid story and/or material to talk about. In my case, an overwhelming majority of my presentations use visual aid like a projector and PowerPoint. Visual aid, if used right, is a powerful tool, and it can also be used to direct a staring (and perhaps sometimes intimidating) audience away from you. However, is that always a good thing?
When you have your presentation set out and you know your format, we come to the more practical preparations. Personally, I like to set up, practice and get a feel for the lecture hall of my presentation beforehand. This way I can make sure that my slides work, I know if there are any pointers available and I can get a 'general feel' for the space that is my stage. However, when it comes to e.g. conferences, this might not be possible, so bring your own gear just in case.

2. Practice

Without visual aid, practice becomes even more important since you can't rely on any slides to help you back on track if you wander off into a dead end. Also, if English isn't your native language it could be even harder than usual to improvise even though you know your story very well.
So practice is key, but it is important to separate practice from learning. Learning your talk is what you do in front of your computer, manuscript in hand perhaps, while practice is what you do 'on stage'. However, when you practice you chose both the setting and the stage. It could be talking to yourself in the mirror or gathering a group of friends as audience in an otherwise empty lecture hall. In preparation of my licentiate seminar i practiced in the actual lecture hall of my licentiate seminar, with and without an audience, every day for two weeks.

3. Feedback

Unfortunately feedback is often overlooked or confused with criticism, and both givers and receivers are responsible for this somewhat lingering mentality.
So from now on, stay away from the word 'criticism' and instead use constructive feedback, both when giving and receiving. Constructive feedback can in turn be either positive or negative, but it's still constructive! Constructive as in a tool for improvement. Because it is important to highlight both that which can be improved and also reinforced.
Feedback could be uncomfortable at first, so bring your friends and close colleagues to your practice talks.
I specifically asked for feedback at three different practice occasions from three different audiences (background, expertise, age, gender, etc.). All their different feedback significantly helped me hone in on the kind of talk and message I wanted to deliver to my intended audience.

4. Consistency

Being a consistently great speaker is unfortunately not just finding your comfort zone and blast through everything you want to say to every audience you will ever have the fortune to stand in front of.
Even a comfortable speaker will have to reflect upon the message being delivered and how to deliver that message. Most, if not all, depend on the audience and the setting. Are you presenting for your peers or a group of school kids? Even if it's an audience of scientists, do they know your field as well as you or your grandmother? Are you the first speaker or the last speaker of the day? Both scenarios might mean that you will have to try extra hard for their undivided attention.
Simplifying and explaining important concepts in detail helps, but don't be too long, and when people start to look tired, change your pace, voice tone and/or body language.

5. Body language

Right here is where your talk will be decided as great or not. Body language. It will not only affect your stance on stage, but it will also affect your voice, your breathing and your connection with the audience.
When I attended the introductory course in research studies in biology during my first year as a PhD student, we learned about something called the 'power spot'. It is not only a certain piece of ground, but also a stance by you, where you are at your most comfortable, confident and connected with your audience. Basically, you stand up front, on your two firm feet, looking the audience in the eye. If you can find your 'power spot', you've already come a long way. The opposite would be standing hunched behind a podium, looking at your own PowerPoint images the entire talk. We've all seen it, and know that it makes people cringe and lose focus.
In your 'power spot' you own the stage and the moment and even though you might be battling your nerves at that very moment, you will still radiate confidence. Time your talk with a few well placed breaks and allow yourself to breathe and the battle will be won. In my last talk I had shorter breaks (just breathe) of a few seconds and then three longer planned breaks where I took the time to zip some water. Trust me, it will help. I asked for specific feedback on this behaviour and everyone I asked thought of it as a sign of confidence or they didn't even notice. Sometime even the audience could use a breather.

6. Patience

After reading this blogpost, even after reading a whole book on the subject or watching a hundred TED talks, there will still be things that could be improved. This list is by no means comprehensive. If you want to be serious about nailing that perfect talk, set up goals along the way. Watch other great speakers and practice different approaches. No speaker woke up one morning, stood up in front of an audience and delivered the most mind-blowing talk ever! It will take time and effort, and likely it's a journey without a final destination.
Personally, I will likely never be fully rid of my nervousness prior to a talk, no matter the context or the audience, but I've learned how to deal with it, and if I compare to where I started I've undoubtedly come a long way. I was on the verge of throwing up prior to talks in high school - where did you start, and where are you now?