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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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Related in: MedlinePlus

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.
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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.

Mentions: We identified the number of unique genes contributed by each cell line to URR by performing the following experiment. Each cell line RNA comprising UHRR, UMRR, and URRR was reverse transcribed to cDNA, labeled with Cy5, and co-hybridized with the corresponding Cy3-labeled UHRR, UMRR or URRR. Stanford 43,000-spot human microarrays, UNC 7,500-spot mouse microarrays, and Agilent 14,500-spot rat microarrays were used for this experiment. Data from 10 human, 11 mouse and 14 rat microarrays were analyzed using GeneTraffic and are presented in Figures 3 through 5. Criteria for gene uniqueness were spot fluorescence intensity greater than 1000 in the Cy5 channel and Cy5/Cy3 ratio greater than 2 in a single cell line. If a spot possessed these same fluorescent intensity characteristics in more than one cell line, it was not considered unique and eliminated from the list of unique genes. The result of this experiment is that each cell line contributes a different number of unique genes to URR. For example, human brain, breast and liver cell lines contribute, respectively, 394, 343 and 335 unique, tissue-specific genes to UHRR (Fig. 3). The total number of highly expressed genes in UHRR contributed by individual cell lines is 2393. Eliminating one or more cell lines from the reference pool would result in reduction of microarray coverage by several hundred unique tissue-specific genes. UMRR includes 1673 uniquely expressed genes out of 7,500 sequences represented on a mouse microarray (Fig. 4) and URRR includes 2205 unique genes out of 14,500 genes on a rat microarray (Fig. 5). These results point out the rationale for pooling RNA from several cell lines to make URR, rather than relying on single cell line RNA.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC394318&req=5

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.
Mentions: We identified the number of unique genes contributed by each cell line to URR by performing the following experiment. Each cell line RNA comprising UHRR, UMRR, and URRR was reverse transcribed to cDNA, labeled with Cy5, and co-hybridized with the corresponding Cy3-labeled UHRR, UMRR or URRR. Stanford 43,000-spot human microarrays, UNC 7,500-spot mouse microarrays, and Agilent 14,500-spot rat microarrays were used for this experiment. Data from 10 human, 11 mouse and 14 rat microarrays were analyzed using GeneTraffic and are presented in Figures 3 through 5. Criteria for gene uniqueness were spot fluorescence intensity greater than 1000 in the Cy5 channel and Cy5/Cy3 ratio greater than 2 in a single cell line. If a spot possessed these same fluorescent intensity characteristics in more than one cell line, it was not considered unique and eliminated from the list of unique genes. The result of this experiment is that each cell line contributes a different number of unique genes to URR. For example, human brain, breast and liver cell lines contribute, respectively, 394, 343 and 335 unique, tissue-specific genes to UHRR (Fig. 3). The total number of highly expressed genes in UHRR contributed by individual cell lines is 2393. Eliminating one or more cell lines from the reference pool would result in reduction of microarray coverage by several hundred unique tissue-specific genes. UMRR includes 1673 uniquely expressed genes out of 7,500 sequences represented on a mouse microarray (Fig. 4) and URRR includes 2205 unique genes out of 14,500 genes on a rat microarray (Fig. 5). These results point out the rationale for pooling RNA from several cell lines to make URR, rather than relying on single cell line RNA.

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