Thermoregulatory Tracing Part II: Probe Synthesis

Once there is a purified sample of DNA, it is time for the next step, probe synthesis.  As I stated before, ISH uses a complimentary RNA probe that binds to the RNA of interest and allows the experimenter to see which cells express that RNA.  In order to visualize the probe, a color reaction must take place.  In this case, the probe contains nucleic acids that are bound to a molecule known as digoxidenin (DIG).  This molecule is incorporated into the probe.  Once the probe has bound to the RNA of interest, the scientist performs a color reaction that involves a colored antibody that binds specifically to DIG, thus lighting up the probes once they are inside the cells.

Producing the probe itself requires several steps.  The first step to probe synthesis is known as linearization.  DNA linearization utilizes restriction enzymes to cut the plasmid in predetermined spots.  These cuts are made specifically so that RNA synthesis will produce a transcript that will bind to the gene of interest based on the direction that RNA polymerization takes place, (since RNA can only be transcribed in the 5-prime to 3-prime direction).

Once the DNA is linearized, RNA synthesis can occur.  RNA synthesis utilizes RNA polymerase sequences that are embedded in certain locations in the plasmid as points at which RNA synthesis can begin.  The correct RNA polymerase is chosen before the reaction in conjunction with the restriction enzymes based on several factors depending on where in the plasmid the scientist wishes to start and which direction the scientist wants the RNA to be synthesized in.  From there, the scientist produces a reaction mix that the linearized DNA will be combined with to produce the probe.  This mix contains the nucleic acids found in RNA, DIG labeled uridine (which will later be important in the color reaction), and the RNA polymerase of choice.

Once you have RNA synthesized, it is time to purify it.  RNA purification is similar to DNA purification in that it takes advantage of the molecular properties of RNA and uses various methods to separate it from the other molecules.  The most important aspect of RNA purification, though, is keeping the RNA on ice, since it is much more volatile and easily degraded than DNA.  Once the purification is complete, it is tested to make sure it is correct and then it is stabilized in a buffer for storage.