Current issues for DNA microarrays: platform comparison, double linear amplification, and universal RNA reference.
DNA microarray technology has been widely used to simultaneously determine the expression levels of thousands of genes. A variety of approaches have been used, both in the implementation of this technology and in the analysis of the large amount of expression data. However, several practical issues still have not been resolved in a satisfactory manner, and among the most critical is the lack of agreement in the results obtained in different array platforms. In this study, we present a comparison of several microarray platforms [Affymetrix oligonucleotide arrays, custom complementary DNA (cDNA) arrays, and custom oligo arrays printed with oligonucleotides from three different sources] as well as analysis of various methods used for microarray target preparation and the reference design. The results indicate that the pairwise correlations of expression levels between platforms are relative low overall but that the log ratios of the highly expressed genes are strongly correlated, especially between Affymetrix and cDNA arrays. The microarray measurements were compared with quantitative real-time-polymerase chain reaction (QRT-PCR) results for 23 genes, and the varying degrees of agreement for each platform were characterized. We have also developed and tested a double amplification method which allows the use of smaller amounts of starting material. The added round of amplification produced reproducible results as compared to the arrays hybridized with single round amplified targets. Finally, the reliability of using a universal RNA reference for two-channel microarrays was tested and the results suggest that comparisons of multiple experimental conditions using the same control can be accurate.
Children's Hospital Informatics Program and Harvard Medical School, 320 Longwood Ave, Boston, MA 02115, USA.
Peter J
PJ
Park
Yun Anna
YA
Cao
Sun Young
SY
Lee
Jong-Woo
JW
Kim
Mi Sook
MS
Chang
Rebecca
R
Hart
Sangdun
S
Choi
D019985:Benchmarking / Q000379:methods / Q000639:trends; D019544:Equipment Failure Analysis / Q000379:methods* / Q000592:standards*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation* / Q000379:methods / Q000592:standards* / Q000639:trends; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods / Q000592:standards* / Q000639:trends; D012313:RNA / Q000235:genetics / Q000592:standards*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards / Q000639:trends; D012680:Sensitivity and Specificity; D013673:Technology Assessment, Biomedical / Q000379:methods / Q000639:trends; D014481:United States
D012313:RNA