Analysis of multiple gene expression array experiments after repetitive hybridizations on nylon membranes.
Nylon membrane-based macroarrays form a widely available alternative to microarrays for the collection of large-scale gene expression data. To carry out repetitive hybridization experiments with nylon cDNA arrays, we used phosphorothioate 33P-cDNA, followed by stripping under relatively mild conditions. We were able to use the same membranes more than 10 times without a measurable reduction in their performance. Thus, our protocol allowsfor more comparative studies of multiple data sets obtained from sequential hybridizations of the same set of membranes. We demonstrate how to analyze repetitive macroarray experiments and to determine the reliability or statistical significance of the gene expression data obtained. Both the averaging of signals per gene and the reversal of nylon membranes had a favorable effect on accuracy. By self-self comparisons, we show that in a duplicate experiment with four membranes, a 2-fold change in the gene expression can be measured reliably.
Hornberg
Jorrit J
JJ
VU Dept of Molecualr Cell Physiology, Amsterdam, The Netherlands.
de Haas
Richard R
RR
Dekker
Henk
H
Lankelma
Jan
J
D001943:Breast Neoplasms / Q000235:genetics*; D015342:DNA Probes; D004730:Endothelium, Vascular / Q000503:physiopathology*; D004867:Equipment Design; D005189:False Positive Reactions; D015870:Gene Expression; D006801:Humans; D008567:Membranes, Artificial; D009757:Nylons; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D010761:Phosphorus Radioisotopes; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014471:Umbilical Veins
D015342:DNA Probes; D008567:Membranes, Artificial; D009757:Nylons; D010761:Phosphorus Radioisotopes