The polymerase chain reaction (PCR) is not the only lab technique that synthesizes new DNA. In my lab, we often need to make DNA from messenger RNA (mRNA) that we extract from cells, in order to see what kinds of mRNAs the cells are making. Normally, cells use DNA to make RNA—this is called transcription. However, a number of enzymes called reverse transcriptases reverse the process of transcription, making DNA from RNA. (Reverse transcriptases are made naturally in small amounts in many plants and animals, but they are most infamously made by retroviruses like HIV.) Though I expect I will perform reverse transcription reaction sometime, I have not yet, and so I am referencing a Harvard University protocol and a Journal of Biological Methods protocol, rather than my own notes.
Step one: mRNA extraction
Starting with a dish growing the cells of interest, the first step is to extract the mRNA. First, work surfaces and equipment should be wiped with RNase ZAP to deactivate any RNases—enzymes that degrade RNA and thus render the mRNA extraction useless. Use a suction pipette to aspirate—suck away—the liquid culture medium. Add phosphate buffered saline (PBS) and swirl to wash the cells, aspirate the PBS, add more PBS, and use a rubber scraper to dislodge the cells that adhere to the culture dish. After adding more PBS and swirling, pipette the cells into a fresh, conical tube. Now, the cells are ready to work with.
Centrifuge the tube at medium speed so that the cells precipitate down to the bottom, forming a pellet. Without disturbing the pellet, aspirate the medium, then add TRIzol (a solution containing phenol), break up the pellet to return the cells into the medium, wait five minutes, add chloroform, shake vigorously, and wait several minutes for the chloroform and phenol to extract the mRNA, DNA, and proteins from the cells.
Centrifuge, this time at very high speed, to separate the cell extracts into several fractions, like separated salad dressing. The mRNA is in the top fraction, so pipette it into a fresh tube without touching the other fractions, which contain unwanted cell components. Centrifuge again at very high speed for a long time so that the mRNA itself precipitates down into a pellet. Carefully remove the liquid above the fragile pellet and wash it with ethanol. Add more ethanol, then break up the pellet with the pipette tip, and, if all went well, the mRNA is finally in solution.
Step two: DNA degradation
Biology is messy, and any purification procedure, like mRNA extraction, is likely to yield an impure product; DNA is likely to be present in the mRNA. To destroy the DNA, which could cause erroneous qPCR results if it were copied during the qPCR mix the following reagents:
DNase buffer 1.5 µL
DNase (an enzyme that degrades DNA, but not RNA) 1.5 µL
mRNA solution (from the mRNA extraction) 12.0 µL
Let the DNase work at room temperature for 15 minutes, then add 1.5 µL of EDTA and heat to 65°C to stop the DNase activity.
Step three: Reverse transcription
The DNA polymerases used to copy DNA during PCR do not copy mRNA. Once the mRNA is purified, it must be converted into DNA by reverse transcription (RT). RT uses the enzyme reverse transcriptase—found in HIV—to synthesize a molecule of DNA from a molecule of mRNA, the reverse direction of normal transcription, in which RNA is made from DNA. First, add 0.5 µL of a primer, then heat to 70°C for ten minutes and place on ice. Then mix the following reagents:
RT buffer (to make sure reverse transcriptase works properly) 25.0 µL
DTT (to protect the DNA) 10.0 µL
dNTP (the monomers that reverse transcriptase links together into DNA) 5.0 µL
RNAsin (to inhibit any potential RNases) 1.25 µL
Superscript II (a solution containing reverse transcriptase) 5.0 µL
Add 9.5 µL of this mixture to each PCR tube containing the purified mRNA. In a thermocycler, heat the PCR tube to 42°C for 90 minutes, then to 50°C and 70°C for ten minutes to allow reverse transcriptase to synthesize the DNA.
Finally, the mRNA has been converted into DNA, which can be used for several purposes, at least one of which—quantitative PCR, which Jinquan has been using a lot recently to measure gene expression—I will describe in a future post.