qPCR – SYBR green to measure a gene

If the genome is a library filled with both useful and useless information, then gene regulation is the librarian who directs you to only the books you find useful. All of the labs on the fourth floor of North Frear are trying to determine how cells find and express—that is, turn on—useful genes while repressing genes that they don’t need at the moment, or ever. Lately, Jinquan has been using a technique called either real-time PCR or quantitative PCR (qPCR) to measure how much of certain genes her cell samples are expressing. When a cell expresses a gene, it first transcribes it, which is the process of making a messenger RNA (mRNA) molecule from the DNA. Thus, if a cell has a lot of mRNA for a certain gene, it is highly expressing that gene.

To measure how much mRNA cells are making for a given gene, we first extract the mRNA from the cell and then synthesize DNA from it, which I described in an earlier post. In order for PCR to work, we need to add primers, which are short sequences of DNA that are complimentary to regions of the DNA strand we want to replicate. This is fortunate, because we only want to measure the DNA of one gene; if every strand was replicated, we would have a lot of DNA, but we wouldn’t know how much of it was for our gene of interest. Since every gene has a different DNA sequence and we know the sequence of the gene that we want to replicate, we can choose a “forward” primer that binds to the beginning of the gene and a “reverse” primer that binds to the end. Only DNA in between the primers (inclusive of the primers) will be replicated.

We add all of the reagents in qPCR to a plastic plate that has 96 wells in it. This is very useful if we want to measure the expression of several different genes, because we can add the primer for one gene to the wells in one column, the primer for a different gene to the wells in another column, and so on. Additionally, we can add DNA from different types of cells to different rows, so that way, we can simultaneously measure the expression of several genes in several types of cells. Here, we measured twelve genes, including the genes for PUMA, MCL1, and actin, in DNA samples from four different kinds of cells.

96-well PCR plate

 

 

 

 

 

 

 

Life technologies: “MicroAmp Optical 96-Well Reaction Plate.” A plate used to perform qPCR.

With 96 wells to fill, it makes sense to make a master mix of reagents common to every tube and then to add the specific reagent to each tube—this saves you 96 tedious pipette actions. Whether the specific reagent is the set of primers or the DNA sample from the type of cell depends on the number of genes and types of cell being measured. If you are measuring more genes than cell types, make master mixes that include the DNA from a specific cell type; otherwise, make master mixes that include the primers for a specific gene. In our case, we had four types of cells and twelve genes. If we made a master mix with the primers for each gene, we would have needed to make twelve different master mixes; instead, we made only four different master mixes by adding the DNA from each cell type to the common reagents.

The common reagents—the DNA polymerase, the free nucleotides, and the all-important SYBR green dye (more on that later)—were pre-mixed for us, so for each DNA sample, we simply added 100 µL of DNA sample to 150 µL of common reagents, making 250 µL total for each DNA sample. Since we had four cell types and 96 wells, each one occupied 24 wells; for each 250 µL mixture, Jinquan pipetted 10 µL into each well, leaving 10 µL as an error margin (no pipette measures perfectly). Then, she added 2 µL of each mixture of forward and reverse primers to each well—twelve different primer sets, eight wells per set.

After adding the reagents, Jinquan sealed the wells closed with a sheet of sticky plastic and placed them in the qPCR thermocycler machine. The thermocycler cycles through several temperatures to allow the DNA polymerase to replicate the DNA. In my earlier post on PCR, I was incorrect about the number of different temperatures used in PCR. The correct cycle is as follows, although specific temperatures and times vary slightly from PCR to PCR:

Temp.             Time                Purpose

94°C                3 min               initially denature: heat to separate the DNA strands from each other

94°C                30 sec              dentature: heat to separate the DNA strands from each other

55°C                30 sec              anneal: cool to let the primers bind to the DNA

72°C                40 sec              elongate: warm to allow DNA polymerase to replicate the DNA

72°C                10 min             finally elongate: allow the DNA polymerase to finish replicating DNA

4°C                  indefinitely      stop: chill for storage of the DNA to prevent DNA breakdown

PCR steps

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Madprime: “PCR,” from Wikimedia Commons. The original DNA template is in blue, primers in red, and newly synthesized DNA in green; the DNA polymerase enzyme that synthesizes new DNA is the green circle.

While the first, penultimate, and last steps are performed once, the bolded steps are repeated for the number of times we want to double the amount of DNA. qPCR usually takes between 30 and 40 cycles. Each cycle, the thermocycler measures the amount of DNA in each well. I mentioned earlier that the master mix contains SYBR green, which is a molecule that binds to DNA. When and only when it is bound, SYBR green will emit green fluorescent light when it is illuminated by blue light. To measure the amount of DNA, then, the thermocycler emits a flash of blue light at the end of the 72°C step, and it measures the amount of green light emitted; more light means more SYBR green is bound to DNA means more DNA.

Below a certain number of DNA molecules, there will be so few SYBR green molecules bound to DNA that the thermocycler cannot detect the green light produced. Therefore, there is a certain threshold number of molecules required for DNA detection. The thermocycler measures the number of cycles that it takes to first detect DNA and calls that number the threshold cycle, abbreviated Ct. Since the amount of DNA doubles every cycle, if one gene reaches threshold one cycle later than another, there were approximately half of the number of DNA molecules to begin with, and thus half of the expression for that gene in the cell that made the DNA. If a gene reaches threshold two cycles later, it had about one fourth of the expression; n cycles later means 1 / 2n times the expression.

qPCR thus provides a way to measure the relative expression of genes in cells, although it does not measure the absolute number of DNA molecules in the original samples. Usually, the expression of each gene is compared to the expression of a reference gene whose expression varies little between different types of cells; actin, a popular choice, is the one which we used in the aforementioned experiment. Comparing the expression of a certain gene to the expression of actin, we can determine how much the cells express it and how the gene is regulated in different types of cells or cells exposed to different chemicals.

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