Biotechnology Engineering

1. Recovery of RNA
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Lecture- 17

2. • Alcohol salt precipitation followed by
centrifugation
• Single step resin binding method
• Silica based column purification
• Glass fiber-based RNA purification
• Isopycnic ultracentrifugation
• RNA isolation kits
• Other methods
Recovery of RNA
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3. Nucleic acids and the salts that drive their
precipitation form complexes with greatly
reduced solubility in ethanol
RNA is efficiently precipitated with 2.5-3 volumes
of ethanol from solutions containing Na+, K+, Li+
or NH4
+ ions.
Precipitated RNA is recovered by centrifugation
at high speed. The resulting pellet is washed with
70% ethanol and air-dried.
Alcohol salt precipitation followed by
centrifugation
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4. • It employs RNA- binding resins (Biotex), and
RNA MATRIXTM (BIO 101)
• It leads to enhancement of the purification of
total RNA
• RNA purified by this method is free from
impurities such as phenol, guanidinium traces
etc.
• No additional carrier protein or density
gradient centrifugation is required
Single step resin binding method
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5. • Nucleic acids adsorb to the solid phase (silica)
depending on the pH and the salt content of
the buffer
Silica based column purification
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6. • Ribopure bacteria kit combines an efficient
glass bead and RNAWIZ mediated disruption
step followed by a glass filter based RNA
purification for high yields of exceptionally
pure bacterial RNA
Glass Fiber-based RNA Purification
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7. • RNA can be isolated by ultracentrifugation
using cesium chloride (CsCl)/ cesium
trifluoroacetate (CsTFA).
• CsTFA is a highly soluble salt that solubilizes
and dissociates proteins from nucleic acid
without the use of detergents
• CsTFA is an excellent inhibitor of RNase
• It shows greater solubility in ethanol
• This method is used to extract ultrapure RNA
Isopycnic Ultracentrifugation
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8. • RNA Isolation kits
• Magnetic bead technology
• Anion exchange chromatography
• Dialysis
Other methods
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