John M. S. Bartlett.pdf - Bio-Nica.info

AU-Differential Display 229
Fig. 1. Schematic representation of AU-DD. (1) Retrotranscription. Dotted line represents
mRNA template. The RNA molecule at the top contains an AU-rich sequence (AU) and a poly-A
tail. G-AU2TTTTTV represents a generic RT primer, with a 5′ anchor of rich on Guanosines,
an AU2 sequence and an oligo(dT). (2) First round of PCR with a single primer GNAU2. (3)
Final products of AU-DD amplification.
7. Precipitate by mixing with 1 volume of isopropanol. Incubate 1 h at –20°C and centrifuge
30 min at 10,000g. Wash with 75% ethanol.
8. Dissolve pellets in 4 M guanidinium thiocyanate. Use at least 0.2 volumes of the lysis
solution used initially. Heat if necessary (pulses of 5 min at 60°C) until there are
not pellet particles left in solution. Be careful because the pellet particles become
transparent and it is difficult to see them. Coprecipitant may be incorporated here adding
1 µL of glycogen (20 mg/mL) and mix.
9. Repeat the precipitation step with isopropanol (9).
10. Wash with 75% ethanol. Dissolve in ultrapure water. The volume of water depends on the
RNA concentration desired. Typically, 50 µL is appropriate to run out all the following
experiments.
11. Total RNA concentration is measured by spectrophotometry and gel electrophoresis. Not
less than 3 µg (see Note 4) or up to 50% of RNA prep is DNase I treated (next step) in a
separate tube, storing the other half as backup or for other purposes at –80°C after mixing
with 3 volumes of 95% ethanol (see Note 5).
12. RNA integrity and concentration is assessed by titration using Escherichia coli rRNAs
(Sigma) as standard in 1% TBE agarose gel electrophoresis and ethidium bromide staining
(13). Different concentrations of commercial E. coli rRNAs (800, 400, 200 ng) are
compared in intensity with different dilutions of the RNA sample.
13. Typically, 5 to 10 µg of total RNA (see Note 6) is incubated at 0.25 µg/µL with DNAse I
for 30 min at 37°C. One microliter is then taken to check the absence of DNA (see
Note 7).
14. RNA is precipitated by adding 0.1 volumes of 3 M KAc, pH 5.0; 1 µL of glycogen; and
3 volumes of 95% ethanol (see Note 8). After standing for 5 min at room temperature,
the pellet is recovered by 10 min of centrifugation in a microcentrifuge at 10,000g and
washed in 75% ethanol (see Note 9).