Universal Reference RNA as a standard for microarray experiments

Abstract

Background: Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location (spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment.

Results: Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97).

Conclusion: Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories.

Figure 1

Microarray experimental design: Comparison of gene expression in two human cell lines using UHRR as a common reference sample. Total RNA isolated from human brain and testis cell lines were reverse-transcribed to cDNA and labeled with Cy5. UHRR was reverse-transcribed to reference cDNA and labeled with Cy3. Each Cy5-labeled cDNA was co-hybridized with Cy3-labeled reference cDNA on microarrays and data analyzed as described in Material and Methods.

Figure 2

Gene expression of human brain and testis cell line KIAA0923 protein. Total RNA isolated from brain and testis cell lines were reverse-transcribed to cDNA, labeled with Cy5 and co-hybridized with Cy3-labeled UHRR onto two 43,000-spot cDNA microarrays (Stanford University). Each microarray had five separate spots containing the probes for KIAA0923 protein gene: (A) Comparison of Cy5 absolute fluorescence intensities registered on five KIAA0923 spots on arrays co-hybridized with Cy5-labeled brain and testis cDNA, and Cy3-labeled UHRR; (B) Comparison of Cy3 absolute fluorescence intensities registered on five KIAA0923 spots on arrays co-hybridized with Cy5-labeled brain and testis cDNA, and Cy3-labeled UHRR; (C) Ratios of intensity values in red and green channels (Cy5/Cy3 ratio); (D) Log2 of the Cy5/Cy3 ratios.

Figure 3

Unique genes contributed by individual cell lines to UHRR. Total RNA isolated from 10 individual human cell lines were reverse-transcribed to cDNA, labeled with Cy5 and co-hybridized with Cy3-labeled UHRR onto 43,000-spot cDNA microarrays (Stanford University). The data was analyzed using GeneTraffic software. Approximately 6000–8000 spots out of 43,000 (14–18%) were flagged on each microarray and excluded from further analysis. Spots with hybridization signals in Cy5 channel higher than 1000 and with Cy5/Cy3 ratio greater than 2 were collected and the number of spots with these characteristics on only one microarray was determined.

Figure 4

Unique genes contributed by individual cell lines to UMRR. RNA from 11 individual mouse cell lines were reverse-transcribed to cDNA, labeled with Cy5 and co-hybridized with Cy3-labeled UMRR onto 7,500-spot mouse oligo microarrays (UNC). The data was analyzed using GeneTraffic software. 300–1000 spots out of 8,000 were flagged on each microarray and excluded from further analysis. Spots with hybridization signals in Cy5 channel higher than 1000 and with Cy5/Cy3 ratio greater than 2 were collected and the number of spots with these characteristics on only one microarray was determined.

Figure 5

Unique genes contributed by individual cell lines to URRR. RNA from 14 individual rat cell lines were reverse-transcribed to cDNA, labeled with Cy5 and co-hybridized with Cy3-labeled URRR onto 14,000-spot rat cDNA microarrays (Agilent). The data was analyzed using GeneTraffic software. 400–1200 spots out of 14,000 were flagged on each microarray and excluded from further analysis. Spots with hybridization signals in Cy5 channel higher than 1000 and with Cy5/Cy3 ratio greater than 2 were collected and the number of spots with these characteristics on only one microarray was determined.

Figure 6

UHRR batch-to batch comparison. (A) Scatter plot of signal intensities from 12,000 spot human microarray (normalized data obtained using GeneTraffic), using Cy3-labeled UHRR batch 1 co-hybridized with Cy5-labeled UHRR batch 2. (B) Scatter plot of Cy3-labeled UHRR batch 1 co-hybridized with Cy5-labeled UHRR batch 1 to 12,000-spot human microarray.