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Tuesday, October 03, 2006

Me and RNAi

RNAi is too good to be true. We live in an era where we want things now. In science we have created a situation where a young researcher must publish or parish. That doesn't give one much time to go out and make a knock-out mouse. The skills required to make the mouse, along with the resources, puts such a project out of reach for someone who has 4 to 6 years to complete a PhD and get a few publications under his/her belt. Along come RNAi. You can buy the RNA, grow some cells and run whatever asssays you would like to demonstrate what it was you had set out to do.

My first experience with RNAi came when I joined a failing bioinformatics company. They had software that identified proteins in pathways not formerly suspected of being their. Of course, none of the protein I was asked to knock out were identified by the software. I was asked to knock out IL6 because it was a favorite of the new CSO who came from a large company that just ended his project in osteoporosis. My job was to knock-out IL6 in RAW cells. To measure the effect I had to stimulate osteoclastogenisis by adding RANK ligand and wait until the RAW cells formed osteoclasts. Once this occured in the negative control (scrambled RNA) I had several assays. First I had to keep track of cell viability. Second there was an assay for tartrate resistant alkaline phosphatase (TRAP). Lastly, I had a bone pit assay that measured the formation of osteoaclasts from the RAW cell precursors.

As everyone who wears the white lab coat can tell you, you start by checking on several transfection reagents. The best one is measured by the best knock down. As a technician things are getting busy. You also have cell cultures that need to be kept in an incubator. One day however, it all worked. Cell viability was the same for scrambled and my chosen siRNA. The TRAP assay showed no TRAP activity in my siRNA but the usual activity in the scrambled. The plate where the osteoclastogenisis assay (pit assay) was taking place showed osteoclast formation in the scramble siRNA group but none in my siRNA treated cells. Eureka! Every assay had worked out. And I was never able to repeat this work again.

What I later discovered was that TRAP activity (using our assay) produces a bell curve over the course of a 5 day assay. Sometime around day 4 the RAW cells begin to rapidly join together. As this occurs there is an increase in TRAP activity right on the cell surface. This correlates with pit formation. I had a control in my experiment but I could (in my head) justify what had happened by imagining a slight delay in how the osteoclastogenisis occured. It only happened in that one experiment becuase I was never able to repeat the results of the first experiment. Others tried as well and were equally unsuccessful. As a laboratory person who really wanted to know what the hell had happened, I tried over and over. I looked at the cells throughout the five day period that it took for them to become osteoclasts. I let them grow for 7 days, for two weeks! I wanted to know everything about how these cells grew. I started several cultures all at different amounts of cells. Cell density at the beginning was critical for getting osteoclastogenisis. Each new osteoclast that (formed several raw) cells put the nuclei from the individual cells together in one region located on one side. On the other side was where most of the TRAP activity could be seen. A pilus formed and reached out to pull in more cells. In this pilus TRAP activity was high. There was a lot going on.

Back in the offices the scientists were busy trying to save the company from running out of money. They took the data from the first experiment and sold it like RNAi was just one of our little tools that can validate the software. Of course, IL6 was not a target identified by the software but that wasn't the point. The point was that we had a tool that we could use. So we tried. None of the targets were known to be in a pathway which made it added more assays to the mix. As money got low we skipped the assays for the known proteins and measured those in the pathway that we were trying to associate with our knock-out. Nothing worked. The company ran out of money and I had to move out of town because there was no work for me in that area of the country.

I moved up north where the new company I worked for was running RNAi experiments. The CEO was convinced that this was the next big thing and he wasn't going to miss it. They ran the transfection experiments. They struggled as we did with the results. Is the RNA in the cells? Why isn't the knock down lasting forever? When do we measure? What should our measurements be? Should we use flourecent tags and a microscope? What are the most reliable assays. In general, they were left to dream up their own assays to show that it was working. This is not PCR where you are promised more DNA and you get more DNA. This is RNAi where you are promised less protein, and you get... slightly less sometimes for an unknown period of time if you use the right piece of RNA with the right transfection reagent. Whatever. Leave it up to the white lab coat people to show up to the meetings with the proper data to tell the story.

This project is all but dead now at my last company. They have since bought another company who is using siRNA to fight the bird flu. The first thing I saw after they bought the company and the bird flu siRNA license was an advertisement for a scientist who has experience with the bird flu and siRNA. If the technology really worked, you don't need a scientist with expertise. The real work would be done in the cells. You need only set everything in motion. You would need expertise in cell culture and handling dangerous viruses. You would need to know how to set up assays. But Cargo Cults are looking for the proper forms. RNAi is a form for a Cargo Cult. It now has a Nobel prize behind it. Let us now sit back and see the real benifits from RNAi. So far, the benifits have come to companies who sell RNAi research materials. What has come out of the research? Papers. And that is what really matters to the modern day scientist. To the non-scientists/non-cargo cult member, what can we make of these papers? Has anyone done anything yet? It's hard to tell.

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