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Universal Reference RNA as a standard for microarray experiments.

Novoradovskaya N, Whitfield ML, Basehore LS, Novoradovsky A, Pesich R, Usary J, Karaca M, Wong WK, Aprelikova O, Fero M, Perou CM, Botstein D, Braman J - BMC Genomics (2004)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stratagene, 11011 N, Torrey Pines Road, La Jolla, CA 92037, USA. nnovo@stratagene.com

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.

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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.
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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.

Mentions: We illustrate one intended use of URR for two-color microarray experiments in the following example. Gene expression in ten human cell lines was compared using UHRR as a common reference. Each individual cell line RNA was compared to UHRR directly on the same microarray. For simplicity the KIAA0923 gene expression is shown only for two samples, brain and testis (Figure 1), to illustrate an inefficiency of the intensity values and advantages of using Cy5/Cy3 ratios. When a common reference sample is used, the multiple microarray hybridizations can be compared. Total RNA from brain and testis human cell lines were reverse-transcribed into cDNA and labeled with Cy5. UHRR was reverse-transcribed into cDNA and labeled with Cy3. Each experimental Cy5-labeled cDNA was co-hybridized with Cy3-labeled cDNA from UHRR onto 43,000-spot cDNA microarrays (Stanford University). The microarrays were processed and data collected and analyzed as described in the Material and Methods. Ratios of Cy5/Cy3 intensities were compared to each other and differentially expressed genes tabulated. Table 1 and Figure 2 illustrate how UHRR allows interpretation of microarray data that would be misinterpreted by ignoring its use. Five different probes for KIAA0923 protein (UniGene cluster Hs.22587), printed on Stanford 43K arrays, are characterized by a seven to eight fold difference in absolute fluorescence intensities in both Cy5 (red) and Cy3 (green) channels on the same microarray (Fig. 2A and 2B). This difference in fluorescence intensity is due to variable spot geometry and probe concentration (data not shown). Using UHRR in combination with brain and testis cell line RNA and computing the Cy5/Cy3 ratio minimizes spot-to-spot variability and allows characterization of high and low gene expression relative to the reference sample. Using Cy5/Cy3 ratios, the data reveals that KIAA0923 gene expression in brain cells is approximately 2-fold higher compared to UHRR (Cy5/Cy3 = 1.57 – 2.74) and 1.5-fold lower in testis cells compared to UHRR (Cy5/Cy3 = 0.54 – 0.87) (Fig. 2C). Logarithmic transformation of the Cy5/Cy3 ratio results in symmetric distribution about zero (Fig. 2D). The transformed data clearly demonstrate expression of the KIAA0923 gene in brain cells and suppression in testis cells.


Universal Reference RNA as a standard for microarray experiments.

Novoradovskaya N, Whitfield ML, Basehore LS, Novoradovsky A, Pesich R, Usary J, Karaca M, Wong WK, Aprelikova O, Fero M, Perou CM, Botstein D, Braman J - BMC Genomics (2004)

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.
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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.
Mentions: We illustrate one intended use of URR for two-color microarray experiments in the following example. Gene expression in ten human cell lines was compared using UHRR as a common reference. Each individual cell line RNA was compared to UHRR directly on the same microarray. For simplicity the KIAA0923 gene expression is shown only for two samples, brain and testis (Figure 1), to illustrate an inefficiency of the intensity values and advantages of using Cy5/Cy3 ratios. When a common reference sample is used, the multiple microarray hybridizations can be compared. Total RNA from brain and testis human cell lines were reverse-transcribed into cDNA and labeled with Cy5. UHRR was reverse-transcribed into cDNA and labeled with Cy3. Each experimental Cy5-labeled cDNA was co-hybridized with Cy3-labeled cDNA from UHRR onto 43,000-spot cDNA microarrays (Stanford University). The microarrays were processed and data collected and analyzed as described in the Material and Methods. Ratios of Cy5/Cy3 intensities were compared to each other and differentially expressed genes tabulated. Table 1 and Figure 2 illustrate how UHRR allows interpretation of microarray data that would be misinterpreted by ignoring its use. Five different probes for KIAA0923 protein (UniGene cluster Hs.22587), printed on Stanford 43K arrays, are characterized by a seven to eight fold difference in absolute fluorescence intensities in both Cy5 (red) and Cy3 (green) channels on the same microarray (Fig. 2A and 2B). This difference in fluorescence intensity is due to variable spot geometry and probe concentration (data not shown). Using UHRR in combination with brain and testis cell line RNA and computing the Cy5/Cy3 ratio minimizes spot-to-spot variability and allows characterization of high and low gene expression relative to the reference sample. Using Cy5/Cy3 ratios, the data reveals that KIAA0923 gene expression in brain cells is approximately 2-fold higher compared to UHRR (Cy5/Cy3 = 1.57 – 2.74) and 1.5-fold lower in testis cells compared to UHRR (Cy5/Cy3 = 0.54 – 0.87) (Fig. 2C). Logarithmic transformation of the Cy5/Cy3 ratio results in symmetric distribution about zero (Fig. 2D). The transformed data clearly demonstrate expression of the KIAA0923 gene in brain cells and suppression in testis cells.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stratagene, 11011 N, Torrey Pines Road, La Jolla, CA 92037, USA. nnovo@stratagene.com

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.

Show MeSH
Related in: MedlinePlus