You can stay true to tradition and use a radioactive label, or you can also use non-radioactive, fluorescently labelled nucleotides. You can then use a column, which is based on the same principle as the column for PCR cleanup, to separate your labelled, reusable probe from the free nucleotides.įor either of these methods you have a choice of labels. When provided by a manufacturer pre-labelled short oligos can be randomly incorporated across the length of your probe during a PCR reaction. And can be done one of two ways: 1) You can label a DNA oligo that is complementary to your RNA or 2) You can random label a PCR product homologous to your sequence. Making the probes for your Northern blot is easy as well. Pro tip: For your first experiment, it’s worth transferring your whole gel to the membrane and hybridizing the full length membrane in case your RNA runs differently than your estimate. Instead you can estimate where your mRNA should be relative to the dyes in the gel and to the ribosomal RNAs you will see after staining. You can also use RNA markers, but these are usually not worth the bother as they degrade quickly and run strangely. The position of rRNA blobs, as revealed by the methylene blue, will then give you benchmark for estimation where your mRNA should be. Step 3: Stain.Īfter you have crosslinked, it is safe to stain the membrane with methylene blue. You can also bake your nitrocellulose membranes in an oven, 30 minutes to 2 hours at 80☌, to fix your RNA in place. To do this you need to expose your membrane to UV light and you will have to calibrate your UV lamp – cross linker machines usually have default programs, just like microwaves. Step 2: Transfer & Link.Īfter successfully separating your RNA it is now time to transfer your RNA to a nitrocellulose or nylon membrane, and crosslink it there to prevent your RNA from washing away in subsequent steps. To learn more about how to do these gels, check out the video on Denaturing Urea Polyacrylamide Gel Electrophoresis by Summer et al. To separate your RNA by size a simple TBE – polyacrylamide – urea gel works well. Separate & Transfer Your RNA Step 1: Gel. With basic precautions, such as cleaning the mold from your workbench and wearing gloves, isolating and maintaining the integrity of your RNA is easy. So don’t let the idea of working with RNA scare you. There are numerous RNA isolation kits on the market now, and lots of RNAse inhibitors available. I think that Northern blots got a bad rap from the days when working with RNA was as hard as it got in molecular biology. And the naming tradition just continued with Western blots. So when invented the RNA method was named “Northern blot” as an homage to the original nucleic acid detection method. Northern Blots are done the same way as Southern blots but RNA is detected instead of DNA. A typical Southern blot experiment goes as follows: 1) run a DNA gel, 2) transfer gel contents onto a membrane, 3) hybridize said membrane with radioactive DNA probe, 4) wash off unbound radioactive probe, and 5) detect radioactive probe. Southern blots are a method of detecting DNA and named after the surname of their inventor Ed Southern. Northern Blots are named after their big brother: The Southern blot. However, because of its unpopular nature finding someone that can teach you about how to do a good Northern Blot can be hard. So sometimes Northern Blots are a necessary evil. Yet, qRT-PCR is prone to false positives and negatives, and reviewers may require Northern Blot confirmation of your qRT-PCR results. Northern Blots fell out of favor for two reasons: The perceived difficulty of working with RNA and because most people don’t like working with radioactivity. After all, it is more common these days to detect and quantify RNA with quantitative real time (qRT)-PCR than with Northern Blots. You might think Northern Blots are an old-fashioned technique.
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