Evaluation of Quality-Control Criteria for Microarray Gene Expression Analysis

Abstract

Background: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. Methods:In the present studies we developed qualitycontrol criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A 260 /A 280 ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix ® HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. Results: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip ® system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01).

Novel report of expression and function of CD97 in malignant gliomas: correlation with Wilms tumor 1 expression and glioma cell invasiveness

“…Quality control criteria included cDNA and cRNA synthesis products within median lengths of 2.0 and 3.0 kb, respectively, and 3ʹ/5ʹ ratios close to 1.00 for the housekeeping genes, GAPDH and β-actin. 8 The "significance-score" algorithm (S-score) developed by Dr. Li Zhang was used to produce a score for the comparisons of the expression summaries between cell groups. 48 The Minimum Information About a Microarray Experiment guidelines have been met, and the microarray raw data have been deposited with the National Center for Biotechnology Institute Gene Expression Omnibus-accession number GSE22578.…”

Precision Profiling and Components of Variability Analysis for Affymetrix Microarray Assays Run in a Clinical Context

“…For Affymetrix analyses, the quality of input RNA and the consistency of sample collection and processing methods have been identified as crucial factors. Dumur and colleagues 7 recently described QC criteria for input sample RNA derived from a variety of sources, and similar criteria have been recommended in a recent best practices document from the Tumor Analysis Best Practices Working Group. 8 Once preanalytical variation has been reduced as much as possible, the analytical precision of the assay becomes the single biggest factor in determining what level of biological change can be seen.…”

Differentially Expressed Genes between Early and Advanced Hepatocellular Carcinoma (HCC) as a Potential Tool for Selecting Liver Transplant Recipients

“…With this extensive preliminary conceptual profiling and practical gene array experience, we have proposed a prospective, longitudinal, multicenter study to answer these questions. We are also aware of the limitations that can arise from the rigorous criteria for total RNA quality, cDNA and cRNA synthesis, and hybridization to ensure reproducible and accurate microarray data (24) and the need for multicenter patient contribution to eliminate sample size misleading associations as occurred for patient 20 in this study.…”

“…To be considered for microarray analysis, the RNA samples needed to pass quality control criteria as 28S/18S ratios greater than 1.5 and A260nm/A280nm greater than 2.0. We established a cutoff value of 30% rRNA contribution to the total area under the electropherogram for RNA samples to be considered as intact or undegraded (24).…”

“…Products of cDNA synthesis and IVT were tested using the Agilent 2100 Bionalyzer (cDNA synthesis 1.5 kb < cDNA < 5.0 kb; IVT 1.0 kb < cRNA < 4.5 kb) (24). Finally, the values for a ratio of 3′/5′ for 2 housekeeping genes were checked: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin; ratios no higher than 3.00 were considered acceptable.…”