Saturday, April 27, 2013

Qiagen Finder 45-pathway Cignal array: the good, the bad and the ugly

I am still not sure whether this is going to be a good review or a bad review. It may shake out one way or another as I write it.

We do a lot of signaling as we work on a intracellular signaling scaffold which functions in a lot of different pathways, so I always pay attention to new email from SABiosciences/Qiagen on their new PCR and luciferase arrays. Upon reading about it the Cignal Finder 45-pathway array seemed to provide an awesome way to apply very sensitive luciferase assays to test 45 pathways in a single shot.
It is a 96 well plate with 45 reporters in duplicates and 3 positive and negative controls that you can use to test you drug, siRNA, over expression plasmid on your cell line of choice. The protocol is simple and straightforward: the DNA is in the wells, you add the cells and transfection reagent and the next day you test for luciferase and renilla activity. At $1,600 for a 10 plate pack, plus dual-luciferase assay reagents, it can run around $200-300 per plate, which in triplicates for 2 conditions can be almost $2,000 per experiment. But screening 45-pathways at once could be totally worth it and SABiosciences always run some kind of deal on the plates (and you can use a fraction of the luciferase reagent recommended in the Promega protocol).

We know our protein of interest would really strongly activate NF-kappaB and we have done it with a different reporter, so that was our positive control. The first plate was amazing! There were very clear difference between reporters, the duplicates looked very consistent and a whole bunch of interesting pathways lit up in addition to NF-kappaB in our experimental condition. The dynamic range of response was incredible with some reporters with intensity in the 2,000,000 intensity units and some in the 2,000. And so we embarked in a huge study with over expression and siRNA...and ordered another 10-pack. Qiagen provided us with a handy Excel file for analysis where we just needed to plug the results of luciferase and renilla for each plate and everything would be calculated and plotted for us.

I was intrigued by some of the results and started paying a lot of attention to the intensity measurements, and that is when things started getting ugly. We had noticed that some of the duplicates were asymmetric in intensity when a low intensity reporter was next to a very high intensity one. I had had similar problems in the past with white plates in our luminometer, where we noticed spillover of fluorescence in neighboring wells. Some people say it's the fluorescence passing trough the white walls, others that the detector is far from the plate and the fluorescence spills over from above like a halo. Also instead of looking at the results in the Qiagen spreadsheet where they are in linear format, I analyzed everything in plate format, where I could see which well was above, below, left and right of any particular reporter. That is when half of our hits became uninterpretable because they could simply be due to spillover from the strong hit next door. Qiagen recommended to half the amount of lysate, but that didn't help much as the results were basically identical. So we were stuck with the option of spending $500 per troubleshooting attempt or find some other solution....

They said it must be our luminometer, which is a possibility, but that's the machine we have in the department and their product should work even for older machines. They gave us 2 plates for free for testing, which was nice, but still leaves for $500 per troubleshooting for each one of the other variables (machine, reagents, lysate amount). Right now I am faced with a conundrum: I have some data I believe from the array and some data I do not believe, which I think is due to spillover. I cannot publish the whole array because I would have to explain it is a problematic product and I have requested for a sample of the 6-8 reporters that are iffy, but I was told I had to buy them individually, which I am not inclined to do at this time, since I do not want to waste my grant money to troubleshoot their product for them.

At the same time, their reporters work beautifully and the data we have is very robust, though for us it was more a 30-pathway array than a 45-pathway because of the issues.....I am open to suggestions from other people who may have tried the product and who may have other ideas. Also, I am not entirely satisfied with the way they recommend to analyze the reporters and I'd love input on that also.

Saturday, April 20, 2013

Advance planning: the benefits of the 0, 18 or 60 month plans

As I start the lab I am trying to figure out where things are going to go in the future and come up with a career development plan. The information on career planning out there is varied and I picked a few suggestions I like.

A 60-month plan. I have been reading a great blog called The Professor Is In, which is run by a former college professor turned career advisor for younger academics. Even if you do not hire her for her services, the blog is great and it covers everything from career planning to what to wear for an interview to how to network. The Professor firmly supports a need for a 5-year plan, nicely structured month by month in an Excel spreadsheet where you identify all the deadlines you are planning to meet. This is not your run of the mill 5-year plan where you daydream about where you want to be in 5 years, this is a place where you can write down grant deadlines, conference deadlines, paper deadlines for years in advance and foresee what you are going to do March 15th two years from now. I am a compulsive planner and list maker, so the 5-year plan spreadsheet was very appealing to me. Grant deadlines are similar from year to year and conferences are planned years in advance, so after a few Google searches I had a nice detailed plan for the first 5 years of the lab. While I think this is great, since it forced me to look for a lot of grant sources and integrate my current plan for publications with grant deadlines, none of this may ever happen, as the plan keeps changing on me, so in the framework of a 5-year plan shorter plans may be in order.

An 18-month plan. I just finished reading Sheryl Sandberg's book Lean In. I have been a long-term Sandberg's fan and followed some of her advice, but this is another story for another post. In Lean In she proposes an 18-month plan, which is not as short as 1 year, but not as daunting as 2 years. The goal is to set an 18-month deadline for improvement or career development: you can either want to develop a skill, enter a new field, set a deadline for funding or publication of a particular project, develop a course. In her formulation, this is not as detailed as having a monthly spreadsheet, but it could also be used as a shorter version of the 5-year plan by planning actual deadlines for only one and a half year at a time, while keeping in mind longer terms obligations, such as when you are supposed to teach for a semester or when you are going up for tenure.

A 0 to 6-month plan. Finally there is who says that planning is futile, that your research will change completely and that your deadlines will fluctuate. I have heard from a few friends that within a year of starting your lab, your goals may have changes completely because the science took unexpected turns, and all the detailed experiment you had planned are not happening. Writing a particular grant may be postponed and other interesting deadlined may come up randomly following a friend's suggestion. Maybe a shorter 3-6 month plan is more realistic and you need to just think in the shorter term to hit specific deadlines without feeling the overwhelming weight of the future. Somewhat like being on a diet where losing 5 lbs in the short term is much less daunting than setting out to lose 30 lbs.

All three options have their pros and cons. I respond well to detailed long-term planning, but at the same time, I am very flexible when the plans do not go the way I planned. Someone who gets discouraged if plans are not kept, may find detailed long-term planning depressing and may prefer the 3-6 month timeline. Overall, I think a combination of the three may be possible with a detailed 6-month plan, a looser but definitive 18-month plan and general broad stroke plan between month 19 and 60. Maybe it's time to revise my 5-year plan spreadsheet and see how it goes.

Saturday, April 13, 2013

More advice to a young investigator: lab management tips

As I was preparing to start this new adventure of running a lab, I have been collecting more and moretips post has been very popular, I thought I'd follow up with some of the new advice I have received.
advice from senior investigators, and since the previous

1) DON'T BE A PERFECTIONIST:  To get your first grants you need to show productivity and you just need to publish nice solid papers. Do not hold on to a project until you think it's perfect because it may slow you down too much. In the race against the tenure clock, this may be a good thing to remember.

2) DON'T BE AFRAID TO BRANCH OUT TO FIELDS YOU DO NOT KNOW: I have already talked about the fear of branching out to a new field and I received this piece of advice right when I was afraid. You have no idea what your career path is going to be like and you never know when the next cool project in the lab is coming from. It could be a conversation over drinks at a conference or just because you want to do things with a friend. You prospective and your expertise may just provide the fresh set of eyes the new field needs.

In addition to the more philosophical scientific suggestions, I also received several practical tips, which are really important as I am thinking whom to hire and how to manage money.

3) WHEN YOU INTERVIEW LISTEN TO YOUR GUTS AND WATCH FOR RED FLAGS: Everyone has heard about the tech or postdoc from Hell, the person who can sink tens of thousand of dollars of reagents in a dead end project, or the person who disrupts the mojo in the lab so badly to make everyone upset. Nobody has a sure fire method to avoid finding yourself in that type of situation, but paying close attention to the red flags during the interview and bringing in as many senior and trusted people as possible to judge the candidate can help. And in addition,

4) ALWAYS CHECK REFERENCES BY PHONE: References are very important and they can tell you a lot about a person. Though I've only hired a few people in my life, I have always had very informative conversation with the referees and have made hiring decisions based on their opinion. So I was not surprised when I was getting this type of advice over and over again. Some people will be very candid and give you a prospective on the applicant you could not get any other way.

5) PAY CLOSE ATTENTION TO YOUR NIH EFFORT: This is a very technical one and may only be applicable if you have a career development award, but you have to be very careful of what the restrictions are on your NIH effort. It does not make sense to spend a lot of time and sweat to apply for a lot of NIH grants, if you cannot actually spare the necessary effort, since you may have to give up the grant or part of the grant. You have to figure out with your budget people and if necessary with your Program Officer what you can and cannot do even before you apply.

6) BE WARY OF LARGE SALARIES and 7) HIRE A GOOD LAB MANAGER: Two contrasting schools of thought. Should you or should you not blow a large amount of your personnel budget on a good lab manager. Everyone who went that route absolutely swears by it saying that it was the best thing they ever did. People who did not hire a lab manager, seem perfectly happy and said they like having the extra money. What is a new PI to do?

Saturday, April 6, 2013

Developing a vision and mission statement for your lab

One of my former bosses always said that running a lab is a lot like running a small business: your research is your product and you have to obtain seed funding to get your work started and continued funding to maintain production, you must advertise so that other scientists and the public know and share your research, and you have to hire and motivate the right employees. In line with setting up your culture, you also need a vision and a mission at the beginning. Who are you? What do you do?
A friend once told me that you have to be able to identify yourself in a couple of words, like a superhero name, and that stuck with me. She was Autophagy Girl, I want to be Extracellular Matrix Girl. That should be what other scientists think of you when they are looking for a collaborator or an opinion on something. What is your focus? And what do you want to be known for? When I joined my postdoc lab, my boss had an idea in his head of what he needed me for, which was not exactly aligned with what I wanted to do. It took six months to realign his expectations: "Yes, we can do that, but I really want to do this."

All universities and hospitals have mottos and mission statements and many are launching major rebranding campaign to define how they want to be perceived in the public opinion. I find quite a few labs also have mission statement and I like the idea of incorporating the goals for our culture in the statement. You always start your talks about what you are about, but what do you stand for? We stand for integrity and communication and innovation, and that needs to be made clear to the people wanting to work for me because I do not always remember to state my core values. I know that people learn by example and that you have to live your values yourself, but it also makes sense to communicate it to them clearly, so that everyone knows what the expectations are.

After looking around for a while I found a handy guide on developing a mission statement. Hope it is useful.

Image credit: By Vegas Bleeds Neon (Own work)