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Monday, October 02, 2006

RNAi My Ass

I've talked about this before. RNAi is too good to be true. When we begin to look at the cell we take the route that an astronomer takes, only instead of big we have to think small. We have to think short distances. We have to think about microenvironments within a cell that could be as different from each other as the atmospheres of Mars and Earth. Sometimes however, we just look at the data that comes from an ELISA or RT PCR and we apply it to our whole universe. We don't think about internal standards or other kinds of controls. Sometimes we look at the data we get and say, "good enough", and the theories start flying. One thing you can say about cell biology people, they are creative. It's paid off in the past, but overall we've got a lot of bullshit.

I'm not classifying myself as a scientist these days. I look at scientists as professional people who apply for grants and work in offices. The lab is the last place you'll find a modern day scientist. They do not need to figure out nature. They need to figure out human beings who work on grant committees and nobel prize committees. I believe the laws of evolution apply to the business of science. Those at the top are those who best understand the human mind and how to best manipulate. It also takes some dishonesty when all else fails. To really get into the measurements of DNA to RNA to protein requires hands on experience. You've got to get in the hood and see the cells that you've transfected. You've got to run the assays that measure RNA and protein levels. Modern day scientists simply don't get that close to the biological organisms being tested nor the equipment, chemistry, and software employed in the data collections.

In the case of RNAi, we simply can't measure the activity of a piece of RNA as is appproaches a cell, enters the cell, makes it's way to the microenvironment where a homologous gene is being expressed, and how it breaks up the process. This process, gene expression, is the essence of all life. The measurements we make to measure gene expression are not hard science. Chemists and physicists practice hard science. They have given us Avagadros number, rates of reactions, limiting factors, the effects of heat and pressure, closed and open systems and so on. When you get to the science of gene expression we have yet to get down to the hard questions. We have no way to measure the mols of mRNA made from DNA and how many mols of protein they make. We don't know much about the effects of temperature, pressure, and pH in the microenvironment where gene expression is taking place. How do we change the microenvironment when we add RNA swimming in a transfection reagent?

The use of antisense RNA to interfere with a gene's activity in C. elegans was first utilised by Su Guo and Ken Kemphues to study par-1 ; however, it was reported that control sense RNA also produced a par-1 mutant phenotype (Cell 81: 611-20, 1995). Subsequently, it was discovered by Fire et al. '98 that it is the presence of dsRNA, formed from the annealing of sense and antisense strands present in the in vitro RNA preps, that is responsible for producing the interfering activity. The concept thus, had already been dreamed up. Add RNA and watch the gene product disappear. The small interferring concept was an intuitive idea of throwing an incomplete and unwanted piece of RNA into the machinery that turns RNA into protein. Fire and Mello were at the right place at the right time.

Technology is the application of science. So far, applying this science has not been very successful. Most companies who began using siRNA have ended their programs. Kary Mullis won the Nobel prize in 1993 for dreaming up PCR. There is no denying that when you use PCR, you have more DNA than when you began. When you use RNAi, you've got some serious questions about whether or not you've got less protein. We don't know exactly which piece of the mRNA makes the best siRNA. We have questions about loops, methylation, number of nucleotides, transfections reagents. Each lab will use it's own protein quantitation method. Some will try RT PCR to measure mRNA levels but there is no reproducible method. If you work in the laboratory, you may have seen a knock down effect. You may have had all the controls needed. But do you really know how well it worked? Can you do it again and again and end up with a knock out that has a margin of error that is significantly small? Too many soft areas of molecular biology and biochemistry are being exploited here.

The ability to control gene expression is something known by the cell. We do not yet know how to do it. We have seen an effect by adding pieces of mRNA. What exactly the cell is doing when exposed to the RNA is not well known. We still are limited in our measurements. If we truly could measure accurately AND use RNAi, we would be better off mapping out metabolic pathways. We could learn about turning genes on and off. We could learn about protein functions like cell cycle regulation. Instead we are throwing RNA at genes of well known drug targets and trying to get a new drug on the market. The ability to use RNAi will soon tell the real story of this discovery. It will fizz out. It's not powerful and not worthy of the Nobel prize.

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