Isolating RNA from cells

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Isolating RNA from cells
Break open cells in the presence of
RNAse inhibitors
Isolate RNA
Total RNA
mRNA
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You isolate all the Total RNA and mRNA in a cell
and you run each in a lane of a gel. You stain
the gel so you can see all the RNAs in each lane.
What will you see?
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NORTHERN BLOTS
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What information are we getting here?
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No drug
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Total RNA
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1) Measure transcription (Run-on
experiments). 2) Block transcription and
measure degradation.
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oligo-dT RNA
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Total RNA
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Real-Time qPCR

• What is real-time quantitative PCR?

A PCR-based method to measure the number of
copies of a particular DNA fragment in a given
sample -Amplification products are labeled by a
DNA binding dye or probe chemistry that emit
fluorescent signal when excited. -The signal
strength of the emitted light is directly
proportional to the amount of PCR product in the
reaction -The fluorescence intensity is detected
and recorded every cycle
DNA amplification is monitored as the reaction
occurs
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TaqMan Chemistry During PCR
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Real-Time qPCR vs.Traditional PCR
30-40 more cycles
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Fluorescence Curve

• As in traditional PCR, amplification during
  real-time qPCR occurs in distinct stages
• Initial
• Logarithmic
• Plateau

Logarithmic phase
Plateau phase
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Fluorescence Curve Initial Phase

• Product is generated but fluorescence remains at
  background levels

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Fluorescence Curve Logarithmic Phase

• Fluorescence rises above background levels during
  exponential accumulation of product
• Fluorescence increases in direct proportion to
  the amount of amplified product present

Logarithmic phase
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Fluorescence Curve Plateau Phase

• No significant increase in DNA quantity or
  fluorescence due to the fact that one or more of
  the reaction components has been depleted

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