Comparing the use of Affymetrix to spotted oligonucleotidemicroarrays using two retinal pigment epithelium cell lines

[摘要] Purpose: The present study was designed to compare the resultsobtained from two different microarray platforms: spotted cDNAs using atwo-color system (Clontech, Atlas Glass Human 3.8) and the Affymetrixplatform. We evaluated the internal consistency within each of theplatforms, and compared the results across the two platforms.Methods: RNA was isolated from two retinal pigment epithelial (RPE)cell lines, D407 cells and ARPE19 cells. Each microarray system requiresa specific RNA isolation and target preparation procedure. To comparethe results between the two platforms, the intensity values for eachplatform were standardized and scaled. This allowed for a directcomparison of the data between two very different microarray platforms.Real-time RT-PCR was used as an independent conformation of expressionlevels for selected transcripts. The protein levels for some of thesegenes were determined using a quantitative immunoblot method.Results: First, we compared the transcriptome of the D407 cell lineto itself. Within each of the platforms there was a high degree ofconsistency. However, when the data from the Atlas Glass Human 3.8microarray platform was compared to that of the Affymetrix platformthere was a dramatic lack of agreement. The second step was to comparethe mRNA profile of the ARPE19 cell line to the D407 cell line. Againthere was good agreement within each platform. When the results of theAtlas Glass Human 3.8 platform were compared to the Affymetrix platform,there was a surprising lack of agreement between the two data sets.Real-time RT-PCR was used as independent means of defining RNA levels inthe two cell lines. In general, the real-time RT-PCR results were inbetter agreement with the Affymetrix platform (85%) than the Atlas Glassplatform (33%). In addition, we also examined the levels of 11 proteinsin these two cell lines using a quantitative immunoblot method. Theresults from this protein analysis had a higher degree of concordancewith the results from Affymetrix platform.Conclusions: In both the Atlas Glass Human 3.8 system and theAffymetrix platform, there is a high degree of internal consistency.However, comparisons between the two platforms show a lack of agreement.In general, the real-time RT-PCR confirmed the results on the Affymetrixsystem more often than those from Atlas Glass arrays. However, in bothcases, conformation by an independent method proves to be ofconsiderable value.

[学科分类] 生物化学/生物物理