Question

I'm confused about a process we're doing in the lab. The goal is to compare gene expression between tissues. We start with PCR, then we do bacterial cloning with a vector, then we do PCR again (using the bacterial colonies as PCR product), followed by gel electrophoresis and "nanodropping". We finish by making a "standard curve" and doing qPCR. Can anyone explain the purpose behind some of these steps?

Answer 1

This information may be incomplete, as it's been several years since I've taken a microbiology course, but I hope it helps somewhat.
It sounds like you understand what PCR/bacterial cloning is used for in your description- basically increasing the amount of genetic material you have to work with.
Gel electrophoresis pulls the genetic information across a gradient with a positive charge at one end and a negative charge at the other. Particles are separated based on their size, where the smaller particles (or, in this case, DNA fragments) are pulled further along the gradient. After the fragments have been pulled, they are stained, usually with Methylene blue, and are visible. The closer the genetics (or more closely related the individuals, in the case of a paternity test) the more similar the banding patterns will be.
I've never heard the term nanodropping before, but after some brief internet research, it seems to be referring to placing the different genetic samples in the line of wells at the back part of the electrophoresis gel. If this is correct, nanodropping is just how you're placing your genetic material, probably with a micropipette.
I would think that you'll be doing this with genetic information from a variety of tissues, then graphing them against your "standard graph" to compare the results from each tissue.
Your last step of qPCR is going to tell you how much mRNA you have. Knowing how much mRNA you have will indirectly tell you about gene expression since mRNA carries the genetic information from the nucleus to the ribosomes where the proteins the genes code for are actually manufactured.
When you combine qPCR with reverse transcription, you can quantify both mRNA and non-coding RNA. Comparing the results from the qPCR and the graphed results from your electrophoresis will answer your starting question: the difference in gene expression between tissues.
Hope I made sense. If you have any questions on what I just said (because I said a LOT!) feel free to ask.

Answer 2

Thanks! This is very helpful, but I'm still a little confused. I understand the basic function of PCR (to create lots of copies of a gene), and it sounds like the purpose of running a gel is just to check and make sure that we've actually copied the gene we want rather than something else. But even if we know that we've replicated a particular gene (e.g. IGF), I don't understand how that tells me anything about the original number. The only way it would make sense to me is if the PCR process always multiplied by a specific number. For example, if I ended up with 100,000 copies and I know that PCR copies a single strand 99,000 times, then I would know that I had 1,000 copies of mRNA (or is it cDNA?) to begin with. But it doesn't sound like it works that way. And whether it does or not, why do we need to insert the gene into bacteria colonies? That's the most confusing part for me.

By the way, nanodropping is a process where we put a drop on a machine and it tells us how many grams of genetic information we have per microliter.

Answer 3

Good to know about nanodropping! I've never heard of such a thing.
PCR doubles the amount of genetic information with each cycle. 2 to 4, 4 to 8, 8 to 16, etc, so if you knew how much DNA you put in there in the first place, you could theoretically predict how much genetic information you have. BUT, it sounds like that's what nanodropping is for.
The bacterial colonies perplex me as well. I can think of three plausible explanations off the top of my head: you're growing the bacteria containing the information in colonies so you have more sources of the original genetic information to work with in case something happens (which doesn't immediately make sense if you're doing PCR to increase how much genetic information you have), your professor or teacher wants you to use the colonies just to get another technique under your belt, or the colonies are somehow processing the genetic information in a way that makes it better suited to the procedures you do afterward.
I wish I had a clear answer for you, but as I said, it's been a while!

Answer 4

Truthfully, it doesnt make sense to me that you are cloning them into a bacterial vector and then getting a standard curve. A standard curve should be created straight from the material that the original DNA came from. Like, if you were trying to influence a certain protein to be upregulated or downregulated in a soybean plant. You would create cDNA from that plants material using known primers for that gene, and then do a qPCR. PCR does replicate by a certain number, though. It multiplies by two each time. If you start with one copy of a gene and amplify it using PCR with 10 steps, you will wind up with 1024 copies. If you start with two copies of a gene and amplify using PCR with 10 steps, you will get 2048 copies. So, the band that you run out on a gel will be two times brighter for the sample that you started with two copies. Of course, I am speaking about all of this like we are in a perfect world, which obviously we are not. However, that is the theory behind qPCR. You can extrapolate backwards from a point on the PCR curve to say which sample had more or less DNA to begin with. Now, to bring the cloning vector back in to the conversation and why it doesn't make sense to me to do it before qPCR. Cloning has its own set of variables that you can somewhat quantify such as the percentage of cells that took up the PCR product that you want it to incorporate. There is an error rate to this, just as there is an error rate to everything in the lab. So, if you do this before your qPCR, then you are increasing the error rate of your qPCR, which is already kind of a sensitive process.