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

RNA Arbitrarily Primed PCR 247
8. 5× reverse transcription mixture: 250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl 2 ,
100 mM DTT, 1.0 mM each dNTP, 2.5 µM oligo(dT) 20 , and 100 U M-MLV reverse
transcriptase.
9. 2× RAP-PCR mixture: 20 mM Tris-HCl, pH 8.3, 20 mM KCl, 6 mM MgCl 2 , 0.35 mM each
dNTP, 2 µM each arbitrary primer (see text); 2 µCi [α- 32 P] dCTP, and 0.5 U/µL AmpliTaq
DNA polymerase Stoffel fragment.
10. 2× Klenow reaction mixture: 20 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 15 mM DTT,
0.050 mM dATP, 0.050 mM TTP, 0.050 mM dGTP, 0.018 mM dCTP, 40 U Klenow.
11. 2× terminal transferase buffer: 400 mM potassium cacodylate; 50 mM Tris-HCl, pH 7.2;
500 mg/mL bovine serum albumin, and 3 mM CoCl 2 .
12. Blocking solution: 10 µg/µL oligo (dA) 20 , 10 µg/µL yeast tRNA, 10 µg/µL human Cot-1
DNA.
13. 20× SSC.
14. Human Cot-1 DNA (Invitrogen #15279, Carlsbad, CA).
15. Klenow (New England BioLabs #MO210S, Beverly, MA).
16. 96-well microtiter plates.
17. 96-well PCR amplification plates.
18. Ultraviolet spectrophotometer.
19. Oven for baking slides.
20. Vacuum desiccator for slide storage.
21. Kodak BioMax X-Ray (Kodak #8715187, Rochester, NY).
22. Succinic anhydride (Sigma #S-7626, St. Louis, MO).
23. 1-methyl-2-pyrrolidinone (Sigma #6762, St. Louis, MO).
24. poly-L-lysine (Sigma #P8920, St. Louis, MO).
2.5. Special Equipment
1. PCR amplification machine (GeneAmp ® PCR System 9700, Perkin-Elmer Cetus, Norwalk,
CT).
2. High-speed robotic arrayer (OmniGrid, GeneMachine, San Carlos, CA).
3. Ultraviolet irradiation device (Stratagene, La Jolla, CA).
4. ScanArray 5000 (GSI Lumonics, Billerica, MA).
5. QuantArray analysis software (GSI Lumonics, Billerica, MA).
6. GoldSeal glass slides (Fisher Scientific, Pittsburgh, PA).
Molecular biology grade, RNase free reagents are used. Sterile disposable polypropylene
was used rather than glassware.
3. Methods
3.1. Preparation of RNA
1. Fibroblasts were grown in culture to about 7 × 10 6 cells per plate and harvested by scraping in
the presence of RTL buffer lysis buffer (RNeasy kit) and homogenized through Qiashredder
columns, both according to the manufacturer’s instructions. Total RNA was isolated using
the RNeasy total RNA purification kit and eluted into 50 µL of distilled water.
2. To 49 µL total RNA was added 6 µL of DNase I digestion buffer, 5 µL of DNase I, and
0.5 µL of RNase inhibitor for 30 min at 37°C.
3. DNase-treated total RNA was purified again using the RNeasy total RNA kit according to
manufacturer’s instructions and eluted into 50 µL of distilled water.
4. RNA concentration was determined by spectrophotometry in TE buffer (10 mM Tris-HCl,
pH 8.4; 0.1 mM EDTA) assuming that one OD unit was equivalent to 40 µg/mL of RNA.