A quality-controlled microarray method for gene expression profiling.
Gene expression profiling on microarrays is widely used to measure the expression of large numbers of genes in a single experiment. Because of the high cost of this method, feasible numbers of replicates are limited, thus impairing the power of statistical analysis. As a step toward reducing technically induced variation, we developed a procedure of sample preparation and analysis that minimizes the number of sample manipulation steps, introduces quality control before array hybridization, and allows recovery of the prepared mRNA for independent validation of results. Sample preparation is based on mRNA separation using oligo(dT) magnetic beads, which are subsequently used for first-strand cDNA synthesis on the beads. cDNA covalently bound to the magnetic beads is used as template for second-strand cDNA synthesis, leaving the intact mRNA in solution for further analysis. The quality of the synthesized cDNA can be assessed by quantitative polymerase chain reaction using 3'- and 5'-specific primer pairs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase. Second-strand cDNA is chemically labeled with fluorescent dyes to avoid dye bias in enzymatic labeling reactions. After hybridization of two differently labeled samples to microarray slides, arrays are scanned and images analyzed automatically with high reproducibility. Quantile-normalized data from five biological replica display a coefficient of variation 45% for 90% of profiled genes, allowing detection of twofold changes with false positive and false negative rates of 10% each. We demonstrate successful application of the procedure for expression profiling in plant leaf tissue. However, the method could be easily adapted for samples from animal including human or from microbial origin.
Degenkolbe
Thomas
T
Max-Planck-Institut für Molekulare Pflanzenphysiologie, D-14424 Potsdam, Germany.
Hannah
Matthew A
MA
Freund
Susanne
S
Hincha
Dirk K
DK
Heyer
Arnd G
AG
Köhl
Karin I
KI
D018076:DNA, Complementary / Q000138:chemical synthesis; D004190:Disasters; D020869:Gene Expression Profiling / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D018515:Plant Leaves / Q000235:genetics; D015203:Reproducibility of Results; D011198:Solanum tuberosum / Q000235:genetics
D018076:DNA, Complementary