[spoiler] AAMC OG B/B Passage 2: Question about research method (locating mRNA in cell)

[autumn123]

So my question isn't specific to any AAMC questions, just understanding the method in the passage. In the passage, I know that the scientists are trying to know where the specific mRNAs are located: the plasma membrane or the cytosol. But how could they know that after doing cell fractionation (when the plasma membrane is destroyed)? I don't know how they know which mRNA associate with which location.

I'm confused with the highlighted part below. I tried googling about cell fractionation and real-time PCR and everything but I still don't get it. Is anyone familiar with this technique? Thank you so much!

Here's the rest of the passage but I don't think it's related.

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[FrameshiftAndrew]

Fractionation protocols for subcellular localization are definitely a thing: here's a relatively recent article, plus several biotech companies provide protocols, etc. Density gradient centrifugation is a key concept in those techniques. The thing is, though, that from the point of view of the MCAT, it doesn't really matter. The sentence that you highlighted indicates that this is possible, and the labels "mem" "cyto" and "no RNA" on the bottom of Figure 1 give you enough information to interpret the findings. In a certain sense, for the purposes of answering questions related to this passage, the mechanism of distinguishing cytoplasmic vs. membrane components may as well be magic.

In other words, for experimental passages where the methods go beyond the standard stuff you're expected to have studied (& this, btw, is why I always recommend double-checking w/ the AAMC content outline if there's ever any doubt), you've basically just got to trust the passage that they're describing something reasonable and possible—the MCAT isn't going to psych you out by describing research methods that seem reasonable but aren't, and insofar as they test you on the methods described in the passage, they can do so in one of two ways: (1) directly testing something that you are supposed to have studied (like blotting or gel electrophoresis), or (2) testing whether you can make a reasonable inference based on what they tell you.

 

[Mr.Blackdoc.5]

Real-time PCR was just to make sure the dna plasmid they transfected the cells with are actually being transcribed into mRNA.

yes, subcellular fractionation can get you a plasma membrane compartment. Remember, when you lyse a cell, you don’t actually destroy the plasma membrane. Your lysis buffer contains a detergent (eg np-40) that will dissociate the membrane (separates lipids making up and within the membrane from each other). This is what causes the cytosolic contents to be extracted for subsequent analysis (eg protein by wb). After you lyse a cell, you centrifuge it at the highest speed so all the insoluble material settles to the bottom of a tube (called clarification). If you do a density gradient centrifugation then you can centrifuge at speeds which will allow you to get soluble fractions containing mitochondria, ribosomes, lysosomes, plasma membrane, etc.

Link below should clarify this further

Overview of Cell Fractionation and Organelle Isolation | Thermo Fisher Scientific - US

This article reviews the steps of subcellular isolation and organelle isolation, including methods for extracting membrane, nuclear and mitochondrial proteins.

www.thermofisher.com