BISC 219/F10: RNAi Lab 11: Difference between revisions
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== RT PCR == | ==Series 3 Reverse Genetics- RT PCR == | ||
You are now ready to do the experiment we have been working on over the past few weeks. You have isolated and purified RNA from your RNAi treated and control worms. The first step in determining if the worms that exhibited a phenotype did so because of a diminished about of RNA or something else, is to reverse transcribe (RT) our mRNA into cDNA (copy DNA - DNA that is lacking introns as it was made from processed mRNA). The second step uses the cDNA as a template for a PCR reaction.<br> | You are now ready to do the experiment we have been working on over the past few weeks. You have isolated and purified RNA from your RNAi treated and control worms. The first step in determining if the worms that exhibited a phenotype did so because of a diminished about of RNA or something else, is to reverse transcribe (RT) our mRNA into cDNA (copy DNA - DNA that is lacking introns as it was made from processed mRNA). The second step uses the cDNA as a template for a PCR reaction.<br> | ||
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Revision as of 09:59, 2 September 2010
Series 3 Reverse Genetics- RT PCR
You are now ready to do the experiment we have been working on over the past few weeks. You have isolated and purified RNA from your RNAi treated and control worms. The first step in determining if the worms that exhibited a phenotype did so because of a diminished about of RNA or something else, is to reverse transcribe (RT) our mRNA into cDNA (copy DNA - DNA that is lacking introns as it was made from processed mRNA). The second step uses the cDNA as a template for a PCR reaction.
There are a few ways you can make cDNA from mRNA and they all involve a specific type of primer. You can use 1) a gene specific primer, 2) an oligo(dT)12-18 primer which binds to the polyA tail of mRNA or 3) random hexamer primers which bind to ALL RNA. What your desired outcome is will determine which primers to use. If you want to do multiple different reactions from the same RT reaction then you should consider using the oligo(dT)12-18 primer or the random hexamer primers. The oligo(dT)12-18 primer binds to the polyA tail found on the end of eukaryotic mRNA's. The mRNA comprises only 1-2% of the RNA in the cell. Random hexamers bind to all RNA in the cell and thus are the most non-specific primers to use.
For our reactions we will use the oligo(dT)12-18 primer for the RT reaction.
We will be using 0.2 ml tubes (small PCR tubes) for our reactions.
- RNA
