Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 137

11. Resuspend pellet in autoclaved water.
Notes: Centrifugation steps may be carried out at room tempera-
ture without signifi cant effects on RNA yield or purity.

For short-term storage, resuspended RNA (in RNase-free water)
should be stored at 20°C; for long-term storage, it should be stored
at −80°C. Try not to expose RNA to drastic changes of tempera-
ture. Extracted RNA should be immediately placed on ice or at
4°C, after 1-2 h at 20°C and then in −80°C. We recommend ali-
quotting RNA into several tubes. This will both prevent damage to
the RNA from successive freeze-thaw events, and help to prevent
accidental RNase contamination.

2.3. RNA Storage

2.4. RNA
Quantifi cation
and Purity

RNA concentrations are determined by spectrophotometric mea-
surement (absorbance at 260 nm). A 260 readings should be greater
than 0.15. With a neutral pH, an absorbance of 1 U at 260 nm
corresponds to 44 g of RNA/ml. A6 values in buffer 1 (1.5 nM

Na-acetate, 0.25 mM EDTA, pH 5.5) may be more accurate than
values obtained in water when RNA extraction from human brain.
The ratio of the readings at 260 and 280 nm ( A 260 / A 280 ) provides
an estimate of the purity of RNA. Pure RNA has an A 260 / A 280 of
1.9-2.3 in 10 mM Tris-Cl, pH 7.5 (recommended) ( 34 ).

μ

2.5. RNA Integrity

RNA is a thermodynamically stable molecule, which is, however,
rapidly digested in the presence of the nearly ubiquitous RNase
enzymes. To evaluate the degree of degradation, these methods
have been applied.

The most commonly used method to assess the integrity of total
RNA is to run an aliquot of the RNA sample on a denaturing
agarose gel stained with ethidium bromide (EtBr). Intact total
RNA run on a denaturing gel will have sharp, clear 28S and 18S
ribosomal RNA (rRNA) bands (eukaryotic samples). The 28S
rRNA band should be approximately twice as intense as the 18S rRNA
band. This 2:1 ratio (28S:18S) is a good indication that the
RNA is completely intact.
Partially degraded RNA will have a smeared appearance, will
lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high
quality RNA. Completely degraded RNA will appear as a very low
molecular weight smear. Inclusion of RNA size markers on the gel
will allow the size of any bands or smears to be determined and will
also serve as a good control to ensure the gel was run properly
( 35, 36 ). Since this approach relies on human interpretation of gel
images, it is subjective, hardly comparable from one lab to another,
and the resulting data cannot be processed digitally.

2.5.1. Agarose Gels

The Agilent 2100 bioanalyzer, is a bio-analytical device based on a
combination of microfl uidics, microcapillary electrophoresis and

2.5.2. Agilent 2100
Bioanalyzer