How do we know that transcriptional regulation is

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- How do we know that transcriptional regulation is commonly used? ALL Diff cells have same genome , but we know that transcribe diff RNA? Have diff transcription factors proteins (translate from transcripts). Can look at diff tissues, see which gene being turned on, by seeing which mRNA being generated. - 1 method: RNA/northern blot. Depends on nucleaic acid hybridization. When piece of DNA/RNA/nucleic acid form H-bonds with its complementary strand (Its target). Can be a short piece.small part of DNA/RNA as hybridization probe that is complementray to our sequence of interest. Anneal the probe to our target. See where the tag happens to be. Kinda like PCR. Do gel aelectropheris, separate fragments by size, smaller travels further, - rNA is hard to take care off, kill nucleases, need to do some sterile techniques, etc RNA blots not quite popular anymore. - If dnt care about size, just wanna know its presence, just spot RNA onto the blot/spot itself, and expose the spot to hybridization. If dot present, thn successful? - RT-PCR . uses RNA as a template instead of DNA. pcr starting off from rna template. Make cDNA copy from rna!! Design pcr primers that recoqgnize signal of interest, not using a probe. Run on gel then detect it. mRNA all ends with poly A tail. Can just use the oligo-dT, for it to anneal, and thn form cDNA. Need to have primer with a specific size, If just 4 bp, more chances for it to anneal somewhere else, not good! Need to be 18-24 bp long, longer, less likely for it to anneal elsewhr. - Reverse transcriptase=retroviruses. DNA polymerase that works with a RNA template!! - Ans: the primers used in RT-PCR. Pcr primers define the region that’s goning to be amplified!! Size of amplified fragment depends on size of primers. Will get more bands if we have all 4 primers (mispipette etc). - RT-PCR much easier to work with compared to using RNA blot. But not quantitative. Solution is using quantitative(real time) PCR. Include a probe with a reporter. Amount of fluoresecene that generated is the amount of DNA regenerated. At some point, fluorescence will reach the threshold level, until it’s maxed out, outside the detection range. CT=the threshold cycle, depends on how much template we begin with. More template=more amplification. Ct1: pass the threshold at 25 cycles, and Ct2 pass the threshold at 28 cycles. Q: which sample contains more copies of template DNA? Sample 1 since requires less cycle to reach the threshold. - 3 doublings, 8 copies more of template? Sample1/sample2 - RT-PCR real time PCR more sensitive, than RNA blot. - In situ hybridization: do directly on tissue itself, kinda like RNA blot. Only shows that it’s there, doesn’t tell you about the amount. - Ex. Use flowers, scan the flower using SEM. Take a cross section of the flower, and see on light microscopy. If probe agamous, we can see darker spots. Washing step (those that didn’t hybridize). Image will show where that label (can be hybridized).
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- Q: compare expression levels=amount of mRNA. Use QUANTITATIVE RT-PCR!! Becz it/s more sensitive. What if looking at quantity of microRNA? Real small amount of nucleotide. USE RNA blot!! Cant use quantitative because it’s not long enough. A good PCR probe is ord 18-24 nucleotide.. so microRNA to small to use RT-PCR.
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