Techniques for Analyzing Gene Expression

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  • 0:00 Gene Expression
  • 1:57 mRNA Detection
  • 6:16 DNA Detection
  • 8:19 Lesson Summary
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Lesson Transcript
Instructor: Adrianne Baron

Adrianne has taught high school and college biology and has a master's degree in cancer biology.

This lesson covers some of the most commonly used techniques to detect gene expression at the mRNA level. We will also discuss the DNA microarray technique. A brief quiz follows the lesson.

Gene Expression

Do you recall learning at some point that we each started out as a single cell that kept dividing? You remember how it goes. We start out as a single cell that contains DNA on a set of 23 chromosomes. The one cell divides into two cells, the two cells divide to make four cells, the four cells divide to make eight cells, etc…until there are enough cells to form every single required cell in our bodies. This means that the cells in your stomach, brain, and heart all originated from the same single cell. This also means that every single one of those cells has the same exact DNA as well.

You are aware that the DNA in each cell tells the cell what to do. But if all cells have the same DNA, and the DNA tells the cells what to do, then why don't the cells in your heart try to digest food? The answer to this is gene expression. Gene expression is the turning on of DNA for it to tell the cell what to do.

So when DNA is turned on, then the DNA gets converted into mRNA and then into protein. The protein is what the cells understand as directions. The proteins are what tell the cells in your heart to beat or the cells in your stomach to digest food. Only the DNA that creates the needed proteins for a particular type of cell are turned on. The DNA for the other proteins are turned off, and gene expression doesn't happen. This is why your heart cells don't try to digest your blood as it passes through!

Now that you know what gene expression is, you may wonder how we can tell which genes are on and off in the different cells in our bodies. Well, scientists have come up with some techniques that help with detection of gene expression. Most techniques detect the mRNA, or proteins, that are produced from genes. Let's look at the mRNA detection techniques now.

MRNA Detection

The first thing that happens with any mRNA detection technique is that the RNA has to be removed from the cell and separated from all other genetic material. This ensures that we are starting with pure mRNA from expressed genes. Now that we have our mRNA sample, we can use our techniques to determine which genes are expressed.

We should also review another couple of terms that will come up a few times in this lesson. Gel electrophoresis is the agarose gel that separates strands of genetic material based on electrical charge. Agarose is taken from agar, a gelatinous material that comes from red seaweed. The charge on DNA, RNA, and proteins is negative. So, the genetic material will move toward the positive end of the gel.

The other helpful part to gel electrophoresis is that the smaller pieces tend to move faster towards the positive end and therefore go farther down the gel than the larger pieces. This allows us to not only detect the strand but to also get an idea of the size of the strand. The other term is DNA probe, which is a single strand of cDNA that can bind to other strands of DNA or mRNA. On to those techniques now!

The most commonly used technique for determining gene expression by detecting mRNA is a northern blot. This is a technique using a nitrocellulose membrane and DNA probe to find mRNA. The mRNA is run on a gel electrophoresis. Each piece of mRNA will create its own band on the gel electrophoresis. The bands on the gel electrophoresis are transferred onto a nitrocellulose membrane. The membrane is then exposed to DNA probes to check for expression of specific genes. If the DNA probe attaches, then the gene is expressed. If the DNA probe doesn't attach, then the gene is not expressed. Northern blots can detect the expression of one or more genes at the same time.

Another technique is the nuclease protection assay, abbreviated as NPA. This is the assay to detect individual mRNA in a mixture of RNA. In this assay, the mRNA is mixed with the cDNA of the genes that we want to know about. If the mRNA that is complementary to the cDNA is present, then they will bind together. Then enzymes are added to the mixture that breakdown the non-bound pieces of RNA. Now we have just the bound mRNA which is from the gene being expressed.

RT-PCR stands for reverse-transcriptase polymerase chain reaction. This is a technique where mRNA is converted back to DNA and replicated. The way that this helps with detecting gene expression is that only the expressed mRNA will be present to convert back to cDNA. Once it is converted back to cDNA and replicated, then the DNA is run on a gel electrophoresis. The cDNA is analyzed to determine which gene it is. The gene identified is the gene that is expressed by that cell.

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