RNA Isolation (objectives)

1
RNA Isolation (objectives)

• Understand why we isolate RNA
• Know ways of inactivating RNases
• Understand the basic principals in isolating
  total RNA and mRNA

2
Why isolate RNA?

• Northern Blots for looking at gene expression
• As a source for cDNA libraries
• RT-PCR
• Nuclease protection assays

3
Extraction of RNA (important factors)

• All solutions need to be treated to remove RNases
• diethyl pyrocarbonate treatment for non amine
  solutions
• protein inhibitors of RNases (RNasin)
• controlling RNase activity
• all glassware, plasticware, etc needs to be
  treated by backing at 180C for 8hrs (glassware)
  or washed in chloroform (plasticware)
• An alternative treatment is washing in DEPC
  followed by autoclaving
• disposable gloves
• be paranoid
• RNase Zap

4
Extraction of RNA (important factors)

• Tissue source of RNA
• Preservation of RNA integrity
• liquid nitrogen
• RNA later RNA stable in tissue stored for up to
  1 day at 37C, 1mth at 4C and indefinitely at
  -20C.

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RNA Later
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Extraction of RNA (manual)

• Suspension of cells in RNA homogenisation buffer
• NaCl, MgCl2, Tris, Nonidet P-40, DTT, RNase
  Inhibitor
• Add RNA extraction buffer
• Tris pH 8, EDTA, NaCl, SDS, Proteinase K
• 37C/30min
• Phenol/chloroform extraction
• Precipitate RNA
• Resuspend and add RNase free DNase I, RNase
  Inhibitor, MgCl2 and DTT (37C/60min)
• Extract in Phenol/chloroform

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Extraction of RNA (manual)

• Precipitate RNA
• resuspend in TE
• There are other methods which use a guanidine
  thiocyanate which effectively lyses cells and
  inactivated ribonucleases
• Trizol is another type of isolation reagent

8
Extraction of RNA (Column)

• Qiagen and Ambion make column based kits for
  isolation of total RNA
• The cells are disrupted in guanidine thiocyanate.
• The lysate is diluted with ethanol and applied to
  a glass or like RNA binding matrix.
• proteins and other contaminants are removed by
  subsequent washes
• The total RNA is eluted

9
mRNA isolation

• Only about 3 of total RNA is specific message
  (mRNA). Most is 28S and 18S
• eg in 5mg of tissue about 10ug of RNA is present
  of which 0.3 ug is mRNA, a rare message accounts
  for 10fg-10pg in total, and a moderately abundant
  message 300pg
• How can we isolate such small amounts of message?

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mRNA isolation

• We utilise the feature of mRNA, ie the poly A
  tail
• Using a column containing a synthetic
  oligonucleotide oligo(dT) attached
• hybridise the mRNA to the oligo(dT) and wash away
  the remainder
• Elute the mRNA by hot buffer
• Usually we do two rounds of purification