Biomedical Engineering Reference
In-Depth Information
References
Warburg O, Christian W (1941) Biochem Z 310:384
Kalckar HM, Shafran M (1947) J Biol Chem 167:461
Whitaker JR, Granum PR (1980) Anal Biochem 109:155
John RA (1992) In: Eisenthal R, Danson MJ (eds.) Enzyme assays: a practical
approach. IRL Press, Oxford, p 59
Welfle H (1996) In: Holtzhauer M (ed.) Methoden in der Proteinanalytik.
Springer, Berlin, p 100
1.2 Quantitative Determination of Nucleic Acids
1.2.1 S
CHMIDT
and T
HANNHAUSER
DNA, RNA, and Protein
Separation Procedure
This procedure has been developed for quantification of the
three types of macromolecules in tissue extracts, where other
biomolecules are also present. Small dissolved amounts of DNA,
RNA, or protein, especially when no material should be consumed
and no interfering substances are in the solution, may be estimated
by UV photometry, but a discrimination between DNA and RNA is
impossible by reading absorbencies (cf. Protocol 1.2.5).
A
14% perchloric acid (w/v) in ddH
2
O
Solutions/Reagents
B 7% perchloric acid (w/v) in ddH
2
O
C 3% perchloric acid (w/v) in ddH
2
O
D diethylether/ethanol 3:1 (v/v)
E1N KOH
F 5% trichloroacetic acid (TCA) (w/v)
G1N NaOH
Mix A solubilized, aqueous tissue sample with an equal volume
of ice-cold Soln. A, and then add ice-cold Soln. B up to 3.0 ml.
The mixture is left for 10 min in an ice bath. After this centrifuge,
leave the acidic solution at 0
◦
C for 10 min with 4000
×
g. Wash the
pellet three times by resuspension in ice-cold Soln. C and further
centrifugation.
Lipids are removed by twofold extraction with ethanol, followed
by a threefold extraction with Soln. D at 30 - 40
◦
C. Centrifuge the
mixtures for a short period at RT between each extraction. Discard
the supernatants. After complete lipid extraction the residual pellet
is air-dried (Schmidt-Thannhauser powder).
Suspend the dry powder in 0.5 ml of Soln. E at RT. Add 0.5 ml
of Soln. A after 1 h and let the mixture for an additional hour in an
ice bath. After that, centrifuge the mixture for 10 min at 4000
g
and 0
◦
C. The supernatant is used for RNA estimation by the orcin
method (Protocol 1.2.2):
first supernatant
.
Add 1.0 ml of Soln. F to the above pellet and heat the mixture to
90
◦
C for 1 h. It is recommended to close the test tubes with a glass
ball to avoid dryness.
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