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.
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.
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1 comment:
Wow!!! Very detailed description of what you do in lab. It sure made it easier to understand the process. I am very proud of you.
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