To ask questions about the sequence, amount adn what regulates the expression of this mRNA.
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Examples of techniques to study a single mRNA
Northern blotting, RT-PCR, RT quantative PCR, DNA sequencing and analysis
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How do we get DNA/RNA out of cells? The first step
break open cell membranes by SDS or lysosome
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How can we obtain cells from tissue
grind in liquid nitrogen or homogenise
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After lysis, what do you do?
Remove cellular debris, precipitate nucleic acids, and then spin down and resuspend
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What do most labs use to extract total RNA?
Spin column kits
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What is different between old method and spin column kits
the precipitation step is replaced with a column method
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How long does the spin column kit take?
30 minutes
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What are the magnetic beads coated in to elute RNA
OligodT - bind to PolyA Rna
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NORTHERN BLOTTING. what do we do first
To isolate RNA from tissue/cells of choice. You can use total RNA or polyA+ RNA
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What do you do in northern blotting?
Electrophorese RNA sample on a denaturing formadehyde agarose gel
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How much sample do you load in northern blotting?
10-20ug of total RNA per lane for moderately abundant gene, or 2-5ug of polyA+ RNA for a low abundance gene
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What do you do after loading?
Transfer to a nitocellulose type membrane
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What do we need to know before northern blotting is carried out?
Knowledge of a part of the sequence, so a probe can be bound.
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What information can northern blotting give us?
Information on relative expression of an mRNA and the full size of the transcript
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What can we use for a reference gene? Why do we need it?
Housekeeping genes such as actin, GAPDH. Possible to use rRNA as well. To recognise how much sample we had to start with. An internal reference.
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bad things about northern blotting
time consuming, often goes wrong. requires a lot of tissue/cell samples
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RT-PCR. Reverse transcriptase polymerase chain reaction. Why do we need to convert mRNA into cDNA before we perform PCR?
The enzymes don't exist in nature to do PCR on mRNA
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What is RT-PCR used for>
Amplifying specific mRNAs
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What enzyme converts mRNA into cDNA? What does it need?
Reverse transcriptase. works 3' to 5' and needs a short mRNA-DNA hybrid. So we use oligodT or random primers
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What's different about the enzyme reverse transcriptase?
Works in the 3' to 5' direction
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What are added to the mRNA sample in RT-PCR
Primers, spanning a short region.
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What should the pcr product contain? Why?
a restriction enzyme site, so that you can test to make sure you've amplified the correct portion of the gene
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What can RT-PCR be used for. What CANT it be used for?
used to confirm the presence of specific mRNA in human tissues and can work with very small samples. It can't tell you how much, or how long the sample is.
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Other cards in this set
Card 2
Front
Examples of techniques to study a single mRNA
Back
Northern blotting, RT-PCR, RT quantative PCR, DNA sequencing and analysis
Card 3
Front
How do we get DNA/RNA out of cells? The first step
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