Biomedical Engineering Reference
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Fig. 2. RNA qualifi cation.
(A)
Poor quality RNA on the
left
, showing little difference
in 18S and 28S band intensities, broad bands, and smearing between bands. Compare
this with the lane on the
right
, noticing that the 28S intensity is approximately twice
that of the 18S. It should be noted that many laboratories might consider the RNA on
the
left
to be acceptable, whereas this laboratory has had the greatest success using
RNA of the quality on the
right
.
(B)
Good quality total RNA showing the 18S band
at half the intensity of the 28S band and sharp bands. In addition, these two samples
were loaded at approximately equal amounts, which has been confirmed by gel
electrophoresis.
for 15 min at 4°C. Remove supernatant and dry pellet for 2-3 min in SpeedVac
(can air-dry as well).
8. Resuspend pellet in DEPC-treated water. Note, however, that if overdried, RNA
will frequently not dissolve).
9. Place in a 55-60°C water bath for 10 min. Store at -80°C.
3.2.4. RNA Quality Assessment and Quantifi cation
Quantity and relative quality of RNA can be determined spectrophoto-
metrically by diluting an aliquot into TE-buffered water (unbuffered water can
produce spurious results) and measuring the UV absorbance at 260 nm and
280 nm (
see
Note 1
). High-quality RNA, essentially free of DNA and proteins,
should have an A
260
/A
280
ratio of 1.9-2.0 and one can expect to obtain a yield
of 1-1.5
g of total RNA/mg of brain tissue. Although the A
260
/A
280
ratio is
a good relative measure of purity, if the RNA is being prepared for use in a
hybridization array experiment, it is imperative to run an aliquot (0.5-1.0
µ
g)
of the RNA on a denaturing 1% agarose gel to verify the presence of two
distinct bands (representing the predominant 28S and 18S RNA species; they
should be in a concentration ratio of approx 2
1) and minimal degradation
(Fig. 2)
. Although this laboratory routinely employs denaturing agarose
gel electrophoresis for determining quality, new technology from Agilent
µ
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