Monitoring gene expression Flashcards
What are the limitations of reporter genes?
- It’s no longer the actual gene so the expression may be altered
- It cannot quantify the DNA
What is RT-qPCR
It is reverse transcription quantitative PCR.
It uses reverse transcriptase which is RNA-directed DNA polymerase - you use your single stranded RNA to create double-stranded DNA.
It chews us the RNA so it doesn’t contaminate and we are only left with the RNA.
What are random primers?
They don’t focus on a specific region. They bind anywhere.
What is in a buffer?
Cofactors for our enzyme to work properly.
Where does RNA transcriptase come from?
Viruses. Some only have RNA. They need DNA to attack the cell so it will use reverse transcriptase to make DNA and integrate into the host genome and reproduce itself.
What are the uses of RT-qPCR?
COVID tests that turn the COVID viral RNA into DNA, amplify it with viral specific primers, and measure fluorescence as a measure of whether you have COVID or not.
What are the steps for this lab?
Isolate RNA, convert it to cDNA, and then do qPCR (denature, anneal, and elongate), measure fluorescence.
What else binds to the column?
DNA. DNAse chews it up.
How much cDNA do you have?
However much RNA you had.
Which region are we amplifying?
The 18s gene and Actin. It’s highly expressed.
What does a low Ct value means?
That DNA passed the Ct value sooner so it was highly abundant.
High expression = high cDNA = high RNA.
Lower Ct value = higher gene expression.
