Old school versus new school in the lab

I was perusing the feeds of my fellow ScienceBloggers the other night when I came across a post by ERV that really resonated with me. In it, she expounds on the benefits of doing things “old school” in the lab, specifically with respect to having hard evidence to defend oneself if ever accused of scientific misconduct. She has a point, but that’s not why the post caught my attention. I’ve actually been struggling with the conflict between “old school” and “new school” recently.

You see, I’ve recently been in the position where I’ve had to add people to my lab, and in fact the entire staff of my lab has turned over completely in a brief period of time. Such transitions occur from time to time, but this is the first time one this radical has happened in my lab, as I’ve only been a principal investigator since late 1999. The new crew is very different from the old crew in a number of ways. For one thing, their knowledge and talent distribution is very different. My old crew was hard core molecular biology. I’m talking cloning a complete human gene from ten introns, linking them together to form what we liked to cal a “Frankenmolecule” cDNA and liking it; bashing the promoter of that gene and then doing chomatin precipitation assays to cover 20 kb or until we found a regulatory element (whichever came first); measuring the copy number of mRNAs in a single cell; identifying microRNAs; doing confocal microscopy on four different fluorescent probes and then confirming it with multiple co-immunoprecipitation to confirm protein interactions of any colocalications observed hard core. Don’t worry if you don’t understand all that. Those who do understand will know just how hard core (and to some extent old school) I mean, and for the rest of you suffice it to say that I mean really hard core–at least for a surgeon’s lab. (I’m not DrugMonkey or PhysioProf, after all, but I do pretty darned good for a surgeon, if I do say so myself.) The new crew is more cell biology-oriented and has shown what to me are some surprising blind spots when it comes to basic molecular biology. I’m talking not even knowing how to do a good transfection. I realize now that in trying to put together a good new team with different skills that I may have gone too far towards the cell biology end. Fortunately, they’re all pretty smart and should be able to pick up the techniques that need to be picked up. Their new skills will also allow me to do stuff that I couldn’t do before (particularly with regard to vascular biology and studies with pericytes and models of inflammation, for which I didn’t have the expertise before), but I find myself explaining a lot of things that I haven’t had to explain for years. I think I’ll be able to whip them into shape (being able to learn is more important than any specified knowledge base), although none of them will ever match my last post doc for his mad skills at molecular biology and there has been (and will be) some frustration on both sides along the way.

One thing, however, where I have run into intermittent conflict, however, and it has nothing to do with differing skill sets. It started when my new crew bought pre-cast polyacrylamide gels. Older faculty will understand why this may have precipitated my first disagreement with the new crew, as may some graduate students.

First, I’ll try to stop slipping into jargon. Polyacrylamide gels are used to separate proteins in a procedure known as a Western blot. In a Western blot, also known as an immunoblot, is a method used to detect specific proteins using antibodies. First, protein extracts are made from the cells or tissue of interest, and the proteins are denatured (unfolded) by chemical means with strong reducing agents that break a type of bond called disulfide bonds. This eliminates the secondary and tertiary structure, allowing them to be separated purely on the basis of molecular weight when forced to migrate through the gel by placing the gel in an electric field. After the proteins are separated, the gel is then placed on a special membrane, and again using the application of an electrical field the proteins are transferred to the membrane, where they are immobilized. They’re then “probed” by incubation in a solution of an antibody against the protein of interest (the “primary” antibody), which is detected by means of a “secondary” antibody directed at the species-specific part of the primary antibody. It’s one of the most commonly used techniques in any lab that does molecular or cell biology.

And, like ERV’s boss, I guess I’m fairly old school (although not nearly so old school that I insist on CsCl gradient purification of DNA). Biomedical scientists out there will understand what I mean when I say that I’m getting a radiation license because I still think that 32P labeling produces better results when doing gel shifts. I’m so old school that I still like Northern blots.

Now here’s where my old school mentality came into conflict with the new school. Actually making the polyacrylamide gel is a bit nasty, as explained in detail here. Why are they so nasty? In brief, you have take two glass plates, clamp and tape them so that they form a thin space between them into which to poor the liquid that will become the gel. Then you have to mix the correct proportion of acrylamide and bis-acrylamide, add a chemical called ammonia persulfate, and quickly poor the mix into the space between the plates (i.e. “cast” the gel). As you might expect, leaks are not uncommon, and, given how close together the plates are, bubbles can be a maddening annoyance. Sometimes for no apparent reason the gel won’t polymerize at all, necessitating recasting it. Even better, unpolymerized acrylamide is a neurotoxin. Still, casting a good polyacrylamide gel and running a high quality Western represent what I thought to be indispensable skills for any biomedical scientist. Moreover, there have been many advances since I was a graduate student to make casting these gels easier, including clamps and holders that make the possibility of a leak much less compared to the old school way of putting two spacers between two specially cut pieces of glass, taping them together, and hoping like hell it didn’t leak when you poured the acrylamide in and that you didn’t get any bubbles that tapping the plates wouldn’t dislodge. It never occurred to me in my entire career since I poured my first polyacrylamide gel during a student rotation some 25 years ago not to pour my own gels or that anyone else wouldn’t pour their own gels either.

They do, though. Now you can buy precast gels. All you have to do is take them out of the plastic package take a piece of tape from the bottom of them, mount them in the gel apparatus, and you’re good to go. Some even have molecular weight markers built in, making measuring the size of the separated and detected proteins a snap.

So what’s the problem? For one thing, they’re way more expensive than pouring your own. (It just occurred to me that that sounded like “rolling your own,” but I assure you it’s not the same thing.) Of course, this is just the raw materials. Pouring a typical gel can take a half an hour or more before samples can be loaded and the separation begun. If you factor in a technician or postdoc’s paid time such precast gels may be cost effective. Indeed, that’s the argument made for all manner of kits in “new school” molecular biology. You can now buy “master mixes” for PCR that have all the ingredients, including magnesium, nucleotides, salt, buffer, and the enzyme that does the magic that is PCR, Taq polymerase, in them already mixed up. Around the time that I was finishing graduate school, suddenly there were Qiagen kits that make isolating plasmid DNA far easier and less messy than the old-fashioned mini and maxi preps that I used to do as a graduate student. At that time they were so expensive that my thesis advisor kept them in his office, and we had to ask him for them when wanted to use them. (Actually, I love Qiagen kits; they’re so much easier and produce cleaner DNA than mini or maxi preps, albeit at a price of a lower yield.) Buffers are all pre-made and ready to go, no muss, no fuss. There are now kits that let you isolate RNA from cells and tissue without all that messy mucking around with toxic guanidine thiocyanate, phenol, and chloroform. Everything is reduced to an easy-to-follow recipe using kits. Add enzyme A to solution B (don’t worry, you don’t need to know what’s in solution B; it’s proprietary anyway). No more film for detecting the chemiluminescence used to detect proteins in Western blot; we use computer imaging instead. Ditto the detection of radiation from 32P used to label DNA.

You’re just being an old fart, youngsters in the lab might say to me. Maybe so, but I don’t think that entirely explains my misgivings about the proliferation of these kits. Something is lost when a scientist relies too heavily on kits, no matter how convenient they can be. I have a new technician in my lab who had done some graduate school. She’s smart and hard working, but she’d never cast a gel herself except for once under supervision in a class. Otherwise, she used precast gel. She has no clue how to troubleshoot it if a gel doesn’t polymerize or if it doesn’t run right, producing smears instead of nice, tight bands. She’s never had to. Yet in never having learned how to cast a gel, I suspect that she doesn’t fully understand just how the gel works to separate proteins. She doesn’t have that intuitive, visceral understanding gained by fiddling with the components needed to make a polyacrylamide gel and troubleshooting when things go wrong. In science, one of the most important abilities is to be able to differentiate when an experiment isn’t working because the science doesn’t support your hypothesis and when an experiment doesn’t work because of a technical problem or because you’re screwing it up. Kits supposedly make the latter possibility much less likely, but over-reliance on them makes it far more less likely that you’ll be able to identify and fix the problem when it is the latter of the two.

Pre-cast gels are actually a relatively minor thing, though, when it comes to my concerns. If I do lab work again (which I do from time to time), I’d probably use them myself because an hour of my time casting the gel is costing my grant and my university far more than any precast gel costs. In any case, I’ve heard the other arguments in favor of kits as well, perhaps the most common (and compelling) of which is that they make basic techniques easier, freeing researchers to think more about the overall design of their experiments or to get more experiments done in the same amount of time. These arguments also postulate that a theoretical understanding of how a technique works is all that’s necessary. Maybe so, but I don’t always find that argument compelling. For one thing, one’s understanding of the science behind a technique can only be strengthened if one has some practical, physical exposure to the technique and how it is done. For another thing, as I mentioned above, what happens when the kit doesn’t work? For example, what happens if your RNA yields are crappy using a Qiagen column? How do you troubleshoot? Sure, you can read the troubleshooting section of the manual, but what if the problem is something that happened to your sample before you use the kit? If you’ve never seen it done any other way than by a cookbook-like following of a printed protocol written by the manufacturer of a proprietary kit, you’ll probably have a hard time figuring it out. I’ve seen students and even postdocs who’ve had no clue, for instance, how to optimize conditions for PCR other than by fiddling with the temperature cycle. It never occurs to them that the concentration of magnesium can have an enormous effect on the efficiency and specificity of the reaction. They’ve always used a kit with a set concentration of magnesium, fiddled with the temperature cycle, and it usually just worked. When faced with a reaction that’s producing multiple products (seen as multiple bands), they have no idea that lowering the magnesium concentration can help with that every bit as much as increasing the annealing temperature.

Old fart or not, “old school” or not, I’m not totally opposed to kits or “recipe” science. I do, however, have definite ideas about what its role should be. That role should not be for beginners who are just learning science and laboratory technique, at least not as exclusive means of doing lab techniques. Old school techniques are an important learning experience and can do much to help new scientists develop an intuitive understanding of the science behind them, as well as valuable troubleshooting skills. At this stage of a scientists’ career kits are little more than crutches, and at some point the crutch has to be thrown away. However, once a scientist has reached a certain level of proficiency, he or she already has developed that understanding, and kits then become very valuable as a convenient time-saver. Moreover, more senior trainees and postdocs have higher salaries, and if there’s one thing I’ve learned as a PI it’s that time is often more valuable than money.

In the end, I’ve come to the conclusion that it more or less depends on the kit, as some kits are more useful than others, namely ones that produce a better end result. That’s the reason I generally like Qiagen columns and kits. They generally in my experience produce cleaner, higher quality DNA preps than mini preps and maxi preps, and they’re certainly a lot less nasty for isolating RNA than using the old school guanidinium thiocyanate/phenol/chloroform method that I “grew up” with as a scientist and don’t require all sorts of toxic chemicals. On the other hand, I’m less enamored of precast gels, which strike me as a long run for a short slide and are expensive to boot. And I really don’t like “master mixes,” where I don’t control the concentration of every reagent. The bottom line is that I probably lean more towards “old school” when it comes to molecular biology kits, but I do make a fair number of exceptions for particularly useful kits that clearly produce better results than the old way.