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Assessing incomplete deprotection of microarray oligonucleotides in situ.

Dressman HK, Barley-Maloney L, Rowlette LL, Agris PF, Garcia-Blanco MA - Nucleic Acids Res. (2006)

Bottom Line: En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine.This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups.Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained.

View Article: PubMed Central - PubMed

Affiliation: Center for Genome Technology, Institute for Genome Science and Policy, Duke University, Durham, NC, USA.

ABSTRACT
En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine. This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups. To solve this quality control problem, we present a sensitive, specific and facile method to detect these groups in situ on arrays using monoclonal antibodies and existing instrumentation. Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained.

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Related in: MedlinePlus

Protecting groups interfere with array function. (A) A regression analysis of the sum of median signal intensity from differentially protected (0–100% DMT, Bz, Ibu and IprPAC) β4-thymosin oligonucleotides that were hybridized with a cDNA probe. Data points represent triplicate experiments; variation within signal intensities is represented by error bars. (B) A regression analysis of the sum of median signal intensity from differentially protected (0–100% DMT, Bz, Ibu and IprPAC) ribosomal protein L9 oligonucleotides that were hybridized with a cDNA probe. Data points represent triplicate experiments; variation within signal intensities is represented by error bars.
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fig2: Protecting groups interfere with array function. (A) A regression analysis of the sum of median signal intensity from differentially protected (0–100% DMT, Bz, Ibu and IprPAC) β4-thymosin oligonucleotides that were hybridized with a cDNA probe. Data points represent triplicate experiments; variation within signal intensities is represented by error bars. (B) A regression analysis of the sum of median signal intensity from differentially protected (0–100% DMT, Bz, Ibu and IprPAC) ribosomal protein L9 oligonucleotides that were hybridized with a cDNA probe. Data points represent triplicate experiments; variation within signal intensities is represented by error bars.

Mentions: The alteration of medians suggested that protecting groups would interfere with microarray performance. In order to further test this, we measured the mean signal intensity for the same mixtures of oligonucleotide probes (β4-thymosin and ribosomal protein L9). The presence of protected oligonucleotides clearly reduced the sum of the mean signal intensity for both probes and a significant reduction could be seen at low levels of protection (Figure 2A and B). The reduction in signal intensity correlated with aberrations in spot morphology. Whereas we cannot conclude that the DMT capping group was responsible for the observed reduction of signal and change in morphology, we can deduce that incomplete removal of DMT, Bz, Ibu or IprPAC leads to suboptimal microarray performance.


Assessing incomplete deprotection of microarray oligonucleotides in situ.

Dressman HK, Barley-Maloney L, Rowlette LL, Agris PF, Garcia-Blanco MA - Nucleic Acids Res. (2006)

Protecting groups interfere with array function. (A) A regression analysis of the sum of median signal intensity from differentially protected (0–100% DMT, Bz, Ibu and IprPAC) β4-thymosin oligonucleotides that were hybridized with a cDNA probe. Data points represent triplicate experiments; variation within signal intensities is represented by error bars. (B) A regression analysis of the sum of median signal intensity from differentially protected (0–100% DMT, Bz, Ibu and IprPAC) ribosomal protein L9 oligonucleotides that were hybridized with a cDNA probe. Data points represent triplicate experiments; variation within signal intensities is represented by error bars.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636491&req=5

fig2: Protecting groups interfere with array function. (A) A regression analysis of the sum of median signal intensity from differentially protected (0–100% DMT, Bz, Ibu and IprPAC) β4-thymosin oligonucleotides that were hybridized with a cDNA probe. Data points represent triplicate experiments; variation within signal intensities is represented by error bars. (B) A regression analysis of the sum of median signal intensity from differentially protected (0–100% DMT, Bz, Ibu and IprPAC) ribosomal protein L9 oligonucleotides that were hybridized with a cDNA probe. Data points represent triplicate experiments; variation within signal intensities is represented by error bars.
Mentions: The alteration of medians suggested that protecting groups would interfere with microarray performance. In order to further test this, we measured the mean signal intensity for the same mixtures of oligonucleotide probes (β4-thymosin and ribosomal protein L9). The presence of protected oligonucleotides clearly reduced the sum of the mean signal intensity for both probes and a significant reduction could be seen at low levels of protection (Figure 2A and B). The reduction in signal intensity correlated with aberrations in spot morphology. Whereas we cannot conclude that the DMT capping group was responsible for the observed reduction of signal and change in morphology, we can deduce that incomplete removal of DMT, Bz, Ibu or IprPAC leads to suboptimal microarray performance.

Bottom Line: En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine.This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups.Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained.

View Article: PubMed Central - PubMed

Affiliation: Center for Genome Technology, Institute for Genome Science and Policy, Duke University, Durham, NC, USA.

ABSTRACT
En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine. This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups. To solve this quality control problem, we present a sensitive, specific and facile method to detect these groups in situ on arrays using monoclonal antibodies and existing instrumentation. Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained.

Show MeSH
Related in: MedlinePlus