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

228 Dominguez et al.
4. Oligonucleotides used as reverse transcription primers (HPLC purified; Genset, Paris,
France) are listed in Table 1. There are two variants used in independent but complementary
experiments, G7AU2dT and GTGAU2dT.
5. RNase H (Gibco BRL) stored at –20°C.
6. Qiaquick PCR purification kit columns (Qiagen).
7. EE (10 mM EPPS), 0.1 mM EDTA, pH 8.2 adjusted with 0.1 N NaOH was used throughout
as a general elution/dilution solution.
8. Reagents for agarose gel electrophoresis.
9. GAPDH primers (see Table 1).
2.3. AU-DD
1. α- 32 P-dATP at 3000 Ci/mmol/DuPont NEN.
2. Platinum Taq DNA polymerase (Gibco-BRL).
3. PCR buffer 1×: 10 mM Tris-HCl, pH 8.8, 1.5 mM MgCl 2 , 50 mM KCl, 0.1% Triton
X-100.
4. dNTPs at 2 mM.
5. AU-DD primers are listed in Table 1.
6. For confirmation of differential expression by standard PCR, specific primers were
derived from sequences of cloned AU-DD products with the program Oligo v5.0 (National
Biosciences Inc.) to have a Tm of 63 to 65°.
2.4. Gel Electrophoresis
1. S2 GibcoBRL electrophoresis apparatus for 0.8-mm thick gels.
2. 40% (382) Polyacrylamide-bispolyacrylamide (Serva, Germany).
3. TEMED (N,N,N′,N′-tetramethylethylethlenediamine).
4. 10% Ammonium persulfate.
5. Whatman 3 MM paper (gel blotting paper).
6. TBE buffer (89 mM Tris Base, 89 mM boric acid, 1 mM EDTA).
7. 20% methanol-10% acetic acid.
3. Method
A schematic representation of AU-DD method is shown in Fig. 1.
3.1. RNA preparation (see Note 1)
1a. Solid tissue is pulverized in liquid nitrogen with mortar and pestle (see Note 2).
1b. Cultured cells are pelleted at 800g for 5 min at 4°C, and put on ice as dry pellets.
1c. For culture flasks with adherent cells, decant, rinse with ice-cold phosphate-buffered saline
and decant by aspiration.
2. Add GCSMP in a proportion of 2 mL per 50 mg of tissue or 10 7 cells.
3. Homogenize lysates at 25,000 rpm for 30 s with a mechanical homogenizer (Ultraturrax
T25, Ika) to ensure both lysis and a complete DNA shearing. Stand for 2 min at room
temperature (see Note 3).
4. Add 0.4 volumes of CI (chloroformisoamyl alcohol) with respect to the complete lysis
solution—including phenol volume—and shake energetically for 10 s.
5. Pour on a prepacked PLG tube. Empty prepacked PLG tubes are 14-mL PPN round-bottom
centrifuge tubes containing 1.5 mL of PLG-heavy and centrifuged 2 min at 1500g; it
should then be left for 10 min on ice.
6. Spin 10 min at 2000g on bench-top centrifuge with swinging bucket rotor. Save upper
aqueous phase to high speed tubes and discard PLG interphase and bottom phase.