High-Sensitivity Detection of DNA Hybridization on Microarrays Using Resonance Light Scattering

Abstract

The application of resonance light scattering (RLS) particles for high-sensitivity detection of DNA hybridization on cDNA microarrays is demonstrated. Arrays composed of approximately 2000 human genes ("targets") were hybridized with colabeled (Cy3 and biotin) human lung cDNA probes at concentrations ranging from 8.3 ng/microL to 16.7 pg/microL. After hybridization, the arrays were imaged using a fluorescence scanner. The arrays were then treated with 80-nm-diameter gold RLS Particles coated with anti-biotin antibodies and imaged in a white light, CCD-based imaging system. At low probe concentrations, significantly more genes were detected by RLS compared to labeling by Cy3. For example, for hybridizations with a probe concentration of 83.3 pg/microL, approximately 1150 positive genes were detected using RLS compared to approximately 110 positive genes detected with Cy3. In a differential gene expression experiment using human lung and leukemia RNA samples, similar differential expression profiles were obtained for labeling by RLS and fluorescence technologies. The use of RLS Particles is particularly attractive for detection and identification of low-abundance mRNAs and for those applications in which the amount of sample is limited.

Microarray analysis of gene expression following preparation of sterile intestinal “loops” in calves

“…For fluorescence methodology, cDNA from control and sample tissue of each animal was labeled with Cy3 and Cy5, respectively, mixed together, and hybridized on duplicate bovine microarrays (Bovine 7500 series; Pyxis Genomics, USA) (Ball et al 2002;DeRisi et al 1996). For RLS methodology the cDNA from control and sample were also mixed and hybridized on duplicate microarrays (Bao et al 2002). Each experiment was performed with duplicate microarrays to compensate for any dye bias by doing dye-swap experiments for both cy-dye and RLS studies (Tsai et al 2003a).…”

“…Each experiment was performed with duplicate microarrays to compensate for any dye bias by doing dye-swap experiments for both cy-dye and RLS studies (Tsai et al 2003a). These technical replicates were completed by switching the labelling protocol for control and sample RNA (Bao et al 2002). Thus, for each methodology (fluorescence and RLS) six microarrays (three biological and two technical replicates) were analyzed for samples collected at 48 h and 12 d post-surgery.…”

“…Slides labeled with Ag and Au particles were scanned with a prototype 16-bit CCD-based white light illumination system (Invitrogen™ Life Technologies, USA) to obtain RLS images (Bao et al 2002).…”

“…Initial microarray experiments utilized Cy-dye based fluorescence detection methodology and only 20 of 7884 ESTs (0.025%) present on the microarray were found to be significantly changed in "loop" tissue when compared to control tissue. To verify this result we employed RLS, a microarray methodology that has been reported to be a more sensitive method for detection of differential gene expression (Yguerabide and Yguerabide 1998a, b;Bao et al 2002;Francois et al 2003).…”

Positive Charged Polymer as a Probe for DNA Determination by Resonance Light Scattering

“…As an analytical technique, RLS is promising and attractive due to its distinct advantages of high sensitivity, convenient operation, simplicity and inexpensive reagents and speediness. Since Pasternack 1,2 first detected assemblies of porphyrin on DNA by the RLS technique, this technique can be used for the quantification of nucleic acids, [3][4][5][6] proteins, 7 pharmaceutical drugs, 8 metal ions, 9 surfactants, 10 sugars, 11 and so on. There is substantial and growing interest in using conjugated polymers (CPs) as the responsive basis for chemical and biological detection schemes.…”