Protocol 101 Affymetrix Gene Chip analyses

HeLa cell total RNA was prepared according to the manufacturer's recommendation using TRIzol (Stratagene) and dissolved in nuclease-free water (Ambion). For each time point, two 40-^l RNA aliquots containing 34 ^g of total RNA were each combined with 10 ^l of 5 x RNA fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc), heated for 70, 80, or 90 s at 90°C in a thermocycler and immediately chilled. Control reactions were kept on ice. After addition of 1 ^.l of a mixture of five different polyadenylated spike RNAs (DapX, LysX, PheX, ThrX, TrpnX) in staggered concentrations, duplicate control and heat-treated samples were pooled and repurified on RNeasy mini columns (Qiagen), including an on-column DNase I digestion step. RNA was eluted in water and quantified by 0D260 measurement. As analyzed by non-denaturing agarose gel electrophoreses, RNAs subjected to heat fragmentation showed obvious signs of degradation, for example a clearly decreased ratio of the 28S and 18S ribosomal RNA bands, while controls appeared to represent intact RNA.

Synthesis of double strand cDNA was performed in duplicate with 13.5 ^g of total RNA from control or progressively degraded samples as described previously by using an anchored T7-oligo-d(T)21-V primer (5/-GCATTAGCGGCCGCGAAATTAATACGACTCACTATAGGGAGA(T)21V-3// MWG Biotech, Ebersberg, Germany) for first strand synthesis (2, 3). cDNAs were purified by phenol/chloroform/IAA/PLG extraction, precipitated and transcribed for 16 h at 37°C in 50-^l reactions containing 40 mM Tris-HCl pH 8.0, 16 mM MgCl2, 2 mM spermidine, 5 mM DTT, 1.1% PEG 20 000, 4 mM GTP and ATP, 1.4 mM UTP and CTP, 0.6 mM Biotin-11-CTP and Biotin-11-UTP, 40 U pyrophosphatase, 50 U RNase inhibitor, and 1.5 ^g T7 RNA polymerase. Biotinylated cRNAs were purified on RNeasy mini columns (Qiagen, Hilden) and quantified by 0D260 measurement. Gel electrophoretic analysis of cRNA samples revealed a detectable shift to smaller average sizes in degraded samples, as expected (not shown). After heat fragmentation of cRNA samples to an average size of 100 to 200 nucleotides, hybridization of Affymetrix HG-U133A arrays with 10 ^.g of cRNA each from control (n =6) and samples degraded for 70 s (n =2), 80 s (n =2) or 90 s (n =2) was performed, followed by washing, staining, and scanning as recommended by the manufacturer (Affymetrix Expression Analysis Technical Manual, 2000). All 12 arrays used in this study were taken from the same batch (lot no. 3 000 016).

Molecular karyotyping by means of array CGH: linking gene dosage alterations to disease phenotypes

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