At least in the life sciences, research involves an awful lot of trying the same thing over and over again hoping for different results. Albert Einstein said the same thing about insanity. Insanity = science? It could be.
Here is a brief background of the experiment I’m attempting. In our grant we proposed the hypothesis that bending the leg back and forth would cause changes in gene expression in the spinal cord. (The rationale for this…never mind, let’s not go there.) Before I joined the project, a genome microarray of 50,000 genes showed changes in gene expression in 180 genes. (Again, let’s not go into the probability that three rats would have changes in gene expression if you looked at 50,000 genes even if you did NOTHING to them. Like I said, all this was done before I joined the team.)
Microarray results have to be confirmed with quantitative RT-PCR. RT-PCR stands for Reverse Transcriptase-Polymerase Chain Reaction. Basically that means you take a strand of RNA, create a DNA copy, then copy that. When you copy DNA, each cycle doubles the DNA. The “quantitative” part means that a bit of fluorescence labels each DNA molecule and a machine reads how bright the fluorescence is at the end of each cycle. All this is converted back into relative numbers so we can compare the amount of RNA that was in the original samples.
Ideally, in the end we are able to say, “This sample had more of this RNA than that sample did.”
My explanation is probably too complicated for most of my readers to follow and too simple (ie inaccurate) for the biosci readers.
All that is still a long way away for me. As each experiment failed to show any RNA at all, I peeled it back step by step. Frustrated at inconsistent RNA yields, I finally gave up using the procedures that had been used by the team, and ordered stuff I’ve used before successfully. As in, I’ve published on it.
I got good RNA. (Qiagen’s RNeasy. Great stuff.)
I moved on to RT-PCR, just the regular RT-PCR, no “q”. Nothing. I tried different primers. At this point I’m not even trying primers for the genes I’m trying to confirm. I’m just trying to get control primers to work.
I ordered control RNA, control primers, and GAPDH primers. I don’t care about GAPDH, and I don’t want to use GAPDH as an internal standard. I want to use a ribosomal RNA, such as Rpl27, as an internal standard. The reason I ordered GAPDH primers is because that is one thing I know every cell in every rat has. If I can find GAPDH in my rats, then I can focus on what’s wrong with the primers for the genes I care about.
(Sorry for the non-researchers who don’t know what primers are. Don’t worry about it. I think you’ll get the point of this article anyway.)
After repeating an RT-PCR over and over again, it finally happened. I got different results.
That’s right, ladies and gentleman. Three lovely bands. Lane 1 is the new RNA with GAPDH primers. Lane 2 is the old RNA with GAPDH primers. Lane 3 is control template and control primers.
Today I did another RT-PCR, testing 5 Rpl27 primers, GAPDH, and a negative control that had GAPDH primers but no RNA.
I got two, and only two, lovely bands. One in the GAPDH lane, and one in the negative control lane.
This probably isn’t clear to non-scientists. “Negative control” means there is NOT supposed to be a band. Clearly I have offended the Flying Spaghetti Monster.
To be quite honest, I don’t exactly do the same thing over and over again expecting different results. In one of the experiments I suspected one of the pipettors was sometimes malfunctioning. I had been doing this upstairs in the lab that has the thermocyclers. I switched to doing it in my own lab with pipettors that I trust. I checked every time I pipetted to make sure there was an appropriate volume of liquid in the pipet tip. And that it expelled.
Another time I thought the gel was questionable. One of the labs upstairs uses TBE. The other one uses TAE. I had made the gel with TBE, and I suspected that the electrophoresis chamber had TAE in it. I made up new TAE, emptied and rinsed the electrophoresis chamber, made a new gel with the new TAE and filled the chamber with the new TAE.
This latest result, where both my positive and negative control had bands? Although I thought I had been extremely careful, I don’t remember with 100% certainty that I put water and NOT TEMPLATE in the negative control tube. So I’ll do it again…and hope for different results.
On the other hand I’m pretty darn confident now that NONE of the five primer sets for Rpl27 work. I have a couple options. I can try to monkey around with the Mg concentration and the primer concentration. I’ve used all of PrimerQuest’s 5 suggested primer sets, so I could try another primer generator software and see what it comes up with. I can assume the published Rpl27 sequence is wrong, perhaps my rats have a different allele than the published sequence (I don’t know how highly conserved it is, although I’d think fairly conserved since it is a ribosomal RNA), and pick a different gene, like Rpl13. I’ll probably order primer sets for Rpl13, and while waiting for them to arrive I’ll play with the Mg and Rpl27 concentrations.
After I eat my spaghetti and repeat the positive and negative controls.