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

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.

Keywords: Dendrimer.; Hybridization specificity; Oligonucleotide microarrays; PCR; Secondary structure; Sensitivity.

Fig. (1)

A Schematic diagram of part of the plasmid, transcribed mRNA and cDNA labelled by either the dendrimer technology or the modified PCR based approach.

Fig. (2)

Effect of formamide and SSC on hybridization efficiency. Relative percentage of mean signal-background for 33 bases probe of PMUR11against PMUR11 and MMUR11 captures using hybridization solutions of variable amounts of formamide 10%, 20% or 30% and either 5x or 2x SSC salt concentration.

Fig. (3)

Effect of probe length on hybridization efficiency. (A) Relative percentage of mean signal-background for 33 bases probe of PMUR11 against PMUR11, MMUR11, PMUR14, MMUR14, PMUR17 and MMUR17 captures using hybridization solution of 30% formamide and 2xSSC salt concentration. (B) Relative percentage of mean signal-background for 120 bases probe of PMUR11 against PMUR11, MMUR11, PMUR14, MMUR14, PMUR17 and MMUR17 captures using hybridization solution of 10% formamide and 5xSSC salt concentration.

Fig. (4)

Specificity comparison for full length, 330 bases and 120 bases probes labelled with either the dendrimer technology and the modified PCR based method. (A) Relative percentage of mean signal-backround for each of the eight full length transcripts against each unique reporter capture sequence. Transcripts were labelled by the dendrimer technology. (B) Relative percentage of mean signal-backround for each of the eight 330 bases transcripts against each unique reporter capture sequence. Transcripts were labelled by the dendrimer technology. (C) Relative percentage of mean signal-backround for each of the eight 120 bases transcripts against each unique reporter capture sequence. Transcripts were labelled by the modified PCR method.

Fig. (5)

Sensitivity comparison between the FAST^® Frame Cassette and the Hybrislip™. (A) Different amounts (pmoles) of the same target, generated by the modified PCR method are plotted against the mean signal-background (RFU) for hybridization carried out in the FAST^® Frame hybridization chamber. (B) Different amounts (pmoles) of the same target, generated by the modified PCR method, are plotted against the mean signal-background (RFU) for hybridization carried out using the Hybrislip™.