RT-PCR

"Reverse transcription polymerase chain reaction" is a five-word mouthful for something more easily said in four simple words: "turning RNA into DNA." The polymerase chain reaction (PCR) single-tubedly revolutionized molecular biology and changed the pace of research in the field forever. It was such an important development that like many other important scientific techniques (including electron microscopy and centrifugation (here called "disperse systems)), it won the Nobel Prize in 1993 and rightfully so. In short, what PCR does is turn a very small amount of DNA into a lot of DNA, still microscopic of course but enough that scientists can actually work with and manipulate it. Here's a video of the process.

The "reverse transcription" means a lot of what it implies. Just as I said how transcription was turning DNA into RNA, the reverse of that process turns RNA into DNA. Viruses are unique in terms of "living" things (viruses are technically not considered living because they need a host in which to reproduce) because some of them use RNA (single stranded or double stranded) as their genetic blueprint instead of DNA (like we and every other organism in every kingdom of life do). HIV is one such example of an RNA-virus. As suggested, these viruses have the natural ability to turn RNA into DNA, a process they need to do in order to integrate their genetic material into that of their hosts'. To do our research, we borrow the special enzyme they use called "reverse transcriptase." It's actually quite remarkable how many uses scientists have for viruses and their products.

So for RT-PCR, instead of the normal DNA polymerase (an enzyme that joins DNA nucleotides together), we use reverse transcriptase. And since we want to convert all of our RNA into DNA (all of the genes), we use random primers (short sequences of nucleotides that specific a part of the genome to amplify) instead of specific ones (those come into play later when we hone into one gene).

Unlike the laborious process of RNA isolation, RT-PCR is quite simple and is just me making up a mix, aliquoting it into tubes with my RNA, then throwing all those tubes into a machine that does the heating and cooling (cycling) for me according to a program a lab mate set up. All I have to do is come back in a couple hours and boom, collect my DNA. Kinda like magic. Then again, a lot of science seems like magic....

Next up: Real-time PCR!

RNA Isolation

So I left off talking about we artificially reduce protein levels (be reducing levels of the template) and what we hope to see from that. Now, briefly, I'll describe how we can tell those protein levels have gone down to the extent that we want them to.

First step, isolating the total RNA from the cells. Think of this step as a lot like panning for gold. You're standing in the middle of the river, with your pan in hand, and scoop up a good mound of the river bed. In it are pebbles, sand, tiny microorganisms, some plant matter, maybe some pieces of pollution, and of course some tiny nuggets of gold. The quality of the gold doesn't matter right now, you just want everything with Au atoms in it. So how do you separate the yellow stuff from everything else? First you shake the pan rather vigorously, then as more and more of the junk falls out you sift more slowly and carefully, occasionally washing the pan with water. Finally, after shaking and washing, you see clinging to your netting tiny little nuggets of gold that you then store into well sealed containers for later analysis.

And now back to RNA. Much like the example, RNA isolation is a process that ranges from the rough and tumble to soft and gentle. Cells from the nucleofection plate are pipetted up, put into tiny centrifuge tubes, spun down at about 100g until they form a pellet, and are dropped into liquid nitrogen where they are flash frozen. (By the way, liquid N2 remains hands down the coolest reagent in science. Though I've handled it many times now, it never gets old).

After the cells are frozen (in which all the liquid around them gets frozen or evaporated off too), they are resuspended in a solution that breaks up the cells to release all their contents. The tubes are spun again so all the heavy stuff (proteins, etc) sink to the bottom while the light stuff (DNA, RNA), stay at the top. The tubes we use are two-chambered in that the bottom chamber collects eluent that flows down from the top chamber. Near the middle of the top chamber is a special filter to which nucleic acids (DNA and RNA) can cling but nothing else can. This way it is easy to discard everything but the nucleic acids throughout the process.

Once protein and nucleic acids are separated, it's time to get rid of DNA since we're not interested in it. We use a tube that has a specific filter for DNA that traps it while letting RNA through. Keep in mind none of these steps are perfect, but these macromolecules are different enough such that these separation methods really work quite well. To get just RNA, it's a matter of washing with various solvents and spinning the tubes many times in a centrifuge. At the end, we get about 2.5 or more micrograms of RNA (that 2.5 millionths of a gram). Very, very small amounts of material here. Once we've got our RNA suspended in water, we throw into the freezer at -80 C until we're ready for the next step: reverse-transcription polymerase chain reaction, also known as RT-PCR.