Use of bioanalyzer electropherograms for quality control and target evaluation in microarray expression profiling studies of ocular tissues

Abstract

Expression profiling with DNA microarrays has been used to examine the transcriptome of a wide spectrum of vertebrate cells and tissues. The sensitivity and accuracy of the data generated is dependent on the quality and composition of the input RNA. In this report, we examine the quality and array performance of over 200 total RNA samples extracted from ocular tissues and cells that have been processed in a microarray core laboratory over a 7-year period. Total RNA integrity and cRNA target size distribution were assessed using the 2100 Bioanalyzer. We present Affymetrix GeneChip array performance metrics for different ocular samples processed according to a standard microarray assay workflow including several quality control checkpoints. Our review of ocular sample performance in the microarray assay demonstrates the value of considering tissue-specific characteristics in evaluating array data. Specifically, we show that Bioanalyzer electropherograms reveal highly abundant mRNAs in lacrimal gland targets that are correlated with variation in array assay performance. Our results provide useful benchmarks for other gene expression studies of ocular systems.

Fig. 1

Experimental workflow in the microarray core laboratory. Total RNA samples are provided to the microarray core laboratory after consultation with clients on experimental design, array selection, and sample preparation. As illustrated to the right of the flow chart, sample quality is checked prior to target synthesis, targets are assessed prior to microarray hybridization, and array data is examined for individual array performance and data distribution patterns across samples within a project. Sample, target, and array performance data are collected in a laboratory information management system for centralized information tracking and to facilitate comparisons among projects and sample types

Fig. 2

Bioanalyzer electropherograms of microaray targets. Electropherogram traces for approximately 200 ng of cRNA target applied to an RNA Nano Chip were generated on the 2100 Bioanalyzer using the mRNA sizing program. a Human brain control cRNA target; b human cornea epithelial cells; c human choroid endothelial cells; d human retinal endothelial cells; e Rhesus macaque lacrimal gland acinar cells; f Rhesus macaque lacrimal gland

Fig. 3

Target assessment patterns of lacrimal gland samples. Electropherogram patterns for microarray targets prepared from Rhesus macaque lacrimal gland acinar cells. The gray dotted line corresponds to target prepared from high-quality human brain RNA. a Electropherogram traces of the two targets associated with the lowest %P measured in the microarray assay for group of 9 acinar cell samples processsed together; b electropherogram traces of the two targets associated with the highest %P measured in the microarray assay