Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 172

Be aware that guanidine thiocyanate does not irreversibly denature RNases.
RNases may renature when denaturing agents are removed and degrade the
sample.

2. Brinkmann/Eppendorf sells a product called “phase lock gel,” which forms a
stable, gelatinous barrier between the organic and aqueous phases. This allows
an easy aspiration and transfer of the aqueous phase.

3. The protocol provided is for up to 300 mg of tissue. For higher amounts of tissue,
the protocol can be upgraded to larger ultracentrifuge tubes and rotors. See ref. 4
for increased amounts of tissue. Many companies provide RNA purifi cation
systems. These systems are most often based on guanidine thiocyanate methods
(9 , 10) that do not require ultracentrifugation steps. In many instances and for
many applications, these systems work well for purifying RNA from brain tissue.
However, for larger amounts of tissue or for purest types of RNA, we prefer to
use the cesium chloride gradient method.

4. A low A 260 /A 280 ratio may be caused by protein contamination. Perform an
additional phenol-chloroform-isoamyl alcohol (25 1) extraction on the

purifi ed RNA, followed by ethanol precipitation and ethanol wash. Loss of RNA
should be expected. A low A 260 /A 280 ratio may also caused by contamination
of the aqueous phase with the phenol phase. Thus, during the extraction do
not completely remove the aqueous layer. Reextract with chloroform-isoamyl
alcohol (49

24

1) to remove phenol, followed by ethanol precipitation and ethanol
wash. Loss of RNA should be expected. Finally, acidic water used for the
spectrophotometric measurement (we use DEPC-treated distilled water for the
spectrophotometric measurement, and high acidity has turned out to be most
often responsible for low A 260 /A 280 ratios) may cause low A 260 /A 280 ratio. In this
case, use Tris-HCl or phosphate buffer, pH 7.6-8.6, instead of distilled water
for spectrophotometric measurement. See ref. 8 for further information on low
pH and A 260 /A 280 ratios.

5. The integrity of the purifi ed RNA can be determined by denaturing agarose gel
electrophoresis. Prepare a miniformaldehyde gel, load 1 µ g of RNA per lane,

g/mL)-TE bath for 15 min,
destain in TE for 5 min, and visualize under UV light (254 nm) ( Caution:
Ethidium bromide is a carcinogen). You should see two bands, the larger 28S
ribosomal RNA band and the smaller 18S ribosomal RNA band (Fig. 6) . The
ratio of 28S to 18S ribosomal RNAs should be approx 2

run at 70 V for 1 h, stain in ethidium bromide (10 µ

1. If RNA samples
have been partially degraded, the bands will be less sharp and the intensity of
the 28S RNA will be diminished. A total degradation of RNA will result in the
loss of the 28S and 18S RNA bands, and the occurrence of a “smear” of smaller,
degraded size RNA in the lanes.

6. Salt contamination can retard the movement of RNA in the gel. To solve this
problem, use ethanol washes to remove any residual salt from the pellet. Add
70-75% ethanol to the RNA pellet, vortex-mix vigorously, and centrifuge for
10 min to recollect the pellet. Carefully aspirate off the ethanol.