lec17_W11

lec17_W11 - 2. Reverse transcription PCR = RTPCR Sensitive...

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2. Reverse transcription PCR = RTPCR Sensitive and fast Can analyze many samples at once Does not require much sample Real-time version very quantitative and sensitive The basic assumption in RT PCR is that the more copies of RNA you started with, the more product you will wind up with at the end.
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What you need in reaction tube in one step RTPCR: Reverse transcriptase Heat-activated Taq (Taq is bound to an antibody that inhibits its activity until it is heated to 94 degrees. This keeps Taq from doing anything until after reverse transcriptase has worked.) dNTPs Forward and reverse primers specific for one mRNA Buffer and Mg Template RNA
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Steps in RTPCR First step – reverse transcription from reverse primer which binds to specific RNA – this done at about 45-50 degrees C for 20 to 30 min. Second step – heat to 90 degrees – denature DNA-RNA duplex, kill RT enzyme activity, and activate platinum Taq Third step – do usual PCR cycling to amplify product Note that you are only amplifying one mRNA because you are using a gene-specific primer.
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5’ 3’ AAAA mRNA 5’ 5’ 3’ 3’ 3’ 5’ DNA AAAA Reagents: -Reverse Transcriptase -dNTPs -Mg +2 and buffer -Reverse DNA primer First step of RT-PCR – creation of first DNA strand using RT: Heat to for 50°C about 10 min – reverse primer anneals and reverse transcriptase creates cDNA copy of that specific mRNA Reverse primer only binds to target mRNA
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Reagents: -Taq polymerase (“hot start”) -dNTPs -Mg2+ & buffer -Forward DNA primer Second step of RTPCR – creation of second DNA strand: 1. Heat to 94 o C: this deactivates RT, activate Taq; denature RNA:DNA duplex, and degrades RNA 5’ 3’ 5’ DNA DNA 2. Drop temperature to 50 to 60 o C – forward primer anneals, and Taq extends to create second DNA strand 3. Repeat PCR steps and amplify for 30 to 40 cycles.
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How to use RTPCR to measure mRNA levels: If the RNAi worked, there would be fewer copies of the unc-22 mRNA in the treated samples, as compared to the untreated. Let’s assume there are 100 copies of the unc-22 mRNA in 100 ng of total RNA from a control worms, and there were only 20 copies in 100 ng of RNA from the RNAi treated worms – so there 5 times as many copies in the untreated samples. But the amount of
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lec17_W11 - 2. Reverse transcription PCR = RTPCR Sensitive...

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