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Effect of Target Length on Specificity and Sensitivity of Oligonucleotide Microarrays: A Comparison between Dendrimer and Modified PCR based Labelling Methods.

Gibriel A - Open Biochem J (2014)

Bottom Line: DNA microarrays are widely used as end point detectors for gene expression analysis.Here we highlight the importance of choosing the optimum target length that would ensure specificity without compromising sensitivity of the assay.For this, eight plasmids that are identical to each other except for a closely related 23 bp unique reporter (UR) sequence were used to examine the hybridization efficiency for these URs.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Molecular Biology Department, Faculty of Pharmacy, Ahram Canadian University (ACU) ; Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
DNA microarrays are widely used as end point detectors for gene expression analysis. Several methods have been developed for target labelling to enable quantification but without taking target length into consideration. Here we highlight the importance of choosing the optimum target length that would ensure specificity without compromising sensitivity of the assay. For this, eight plasmids that are identical to each other except for a closely related 23 bp unique reporter (UR) sequence were used to examine the hybridization efficiency for these URs. Targets of various lengths were generated and labelled as follows: full length and 330 bases transcripts using a dendrimer labelling method, 120 bp amplicons by the modified PCR end labelling method and synthetic labelled targets of 33 bases. This report also shows the advantages of using the modified PCR method over other labelling methods in generating labelled amplicons of the desired lengths to maximize hybridization efficiency.

No MeSH data available.


A Schematic diagram of part of the plasmid, transcribed mRNA and cDNA labelled by either the dendrimer technology or the modifiedPCR based approach.
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Figure 1: A Schematic diagram of part of the plasmid, transcribed mRNA and cDNA labelled by either the dendrimer technology or the modifiedPCR based approach.

Mentions: In this manuscript, we optimise and compare the specificity and sensitivity of the dendrimer and a modified PCR based labelling method. HEK293 cells were separately transfected with 8 plasmids that are identical except for a unique 23 bp sequence (UR). All these URs are closely related with a degree of identity ranging from 35% to 74% (Supplementary information, Table S1). Multiple mismatches are distributed over the entire UR sequences so that each UR can act as a mismatch to the remaining ones (Supplementary information, Fig. (S2)). Neither a dA tail nor dT primer was used in the modified PCR method in order to preserve RNA transcript relative abundance. Instead, a universal primer pair was used to generate 120 bp short amplicons of equal size for all transcripts. Full length and 330 bases transcripts generated using the Genisphere oligo dT and customized antisense primers respectively Fig. (1) were used to assess the dendrimer technology.


Effect of Target Length on Specificity and Sensitivity of Oligonucleotide Microarrays: A Comparison between Dendrimer and Modified PCR based Labelling Methods.

Gibriel A - Open Biochem J (2014)

A Schematic diagram of part of the plasmid, transcribed mRNA and cDNA labelled by either the dendrimer technology or the modifiedPCR based approach.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927376&req=5

Figure 1: A Schematic diagram of part of the plasmid, transcribed mRNA and cDNA labelled by either the dendrimer technology or the modifiedPCR based approach.
Mentions: In this manuscript, we optimise and compare the specificity and sensitivity of the dendrimer and a modified PCR based labelling method. HEK293 cells were separately transfected with 8 plasmids that are identical except for a unique 23 bp sequence (UR). All these URs are closely related with a degree of identity ranging from 35% to 74% (Supplementary information, Table S1). Multiple mismatches are distributed over the entire UR sequences so that each UR can act as a mismatch to the remaining ones (Supplementary information, Fig. (S2)). Neither a dA tail nor dT primer was used in the modified PCR method in order to preserve RNA transcript relative abundance. Instead, a universal primer pair was used to generate 120 bp short amplicons of equal size for all transcripts. Full length and 330 bases transcripts generated using the Genisphere oligo dT and customized antisense primers respectively Fig. (1) were used to assess the dendrimer technology.

Bottom Line: DNA microarrays are widely used as end point detectors for gene expression analysis.Here we highlight the importance of choosing the optimum target length that would ensure specificity without compromising sensitivity of the assay.For this, eight plasmids that are identical to each other except for a closely related 23 bp unique reporter (UR) sequence were used to examine the hybridization efficiency for these URs.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry and Molecular Biology Department, Faculty of Pharmacy, Ahram Canadian University (ACU) ; Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

ABSTRACT
DNA microarrays are widely used as end point detectors for gene expression analysis. Several methods have been developed for target labelling to enable quantification but without taking target length into consideration. Here we highlight the importance of choosing the optimum target length that would ensure specificity without compromising sensitivity of the assay. For this, eight plasmids that are identical to each other except for a closely related 23 bp unique reporter (UR) sequence were used to examine the hybridization efficiency for these URs. Targets of various lengths were generated and labelled as follows: full length and 330 bases transcripts using a dendrimer labelling method, 120 bp amplicons by the modified PCR end labelling method and synthetic labelled targets of 33 bases. This report also shows the advantages of using the modified PCR method over other labelling methods in generating labelled amplicons of the desired lengths to maximize hybridization efficiency.

No MeSH data available.