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The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters

ABSTRACT: Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest-neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching complementary sequence and other spots with one or two mismatches: in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays. Keywords: experiment to assess technical aspects of hybridization in microarrays

ORGANISM(S): synthetic construct

PROVIDER: GSE14547 | GEO | 2009/03/01

SECONDARY ACCESSION(S): PRJNA111641

REPOSITORIES: GEO

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Project description:Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest-neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching complementary sequence and other spots with one or two mismatches: in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays. Keywords: experiment to assess technical aspects of hybridization in microarrays The study consists of four experiments. Each experiments consists of one slide with eight identical microarrays which were hybridized with an identical solution but at a different concentration. Per experiment a single target sequence in solution was used for hybridization: target1 to target4 (Table 1 main manuscript or description of samples below). Each target oligo was bought in duplicate, referred to as oligos a and b. In experiment 1 and 2 a fragmentation step (f) was applied. More details can be found in the main manuscript especially Table 3.

Project description:Cell culture: H9 hESCs were grown using feeder-free conditions for 7 days after passage then either RNA was harvested or embryoid bodies were prepared, cultured in suspension for 4 days, plated on gelatin-coated plates, and cultured for an additional 9 days in embryoid body medium. Detailed protocols for feeder-free culture and embryoid body formation are available at www.geron.com/scprotocols.pdf RNA preparation: Cells were then harvested in situ by overlaying with RLT lysis buffer (Qiagen, Valencia, CA). RNA was prepared using RNeasy Midi kits as described by the manufacture (Qiagen). To remove contaminants, RNA was precipitated by adding an equal volume of LiCl Precipitation Solution (Ambion, Austin, TX), washed with 70% ethanol and resuspended in water. Probe preparation: Probes were prepared from 20ug of total RNA by direct incorporation of Cy3 or Cy5 dCTP (Amersham Biosciences, Piscataway, NJ) into oligo dT primed first strand cDNA using SuperscriptIII reverse transcriptase (Invitrogen), as described by the dye manufacturer. RNA was removed by alkaline hydrolysis, then neurtralized probes were purified using Qiaquick columns as described by the manufacturer (Qiagen). Hybridization/Washing: Purified probes were dried and resuspended MWG Hybridization Buffer C (MWG Biotech) with the addition of 20 ug of sheared salmon sperm DNA. Slides were hybridized overnight at 42°. After the coverslips were removed, the slides were rinsed in 2X SSC + 0.1% SDS, washed in 2X SSC + 0.1% SDS for 5 min, 1X SSC for 5 min, 0.2X SSC for 5 min and 0.1X SSC for 5 min. Slides were then dried by centrifugation. Scanning: Arrays were scanned using a GenePix 4000A microarray scanner (Axon Insturments, Fremont, CA) with 100% scan power and PMT voltage set to minimize saturated pixels and approximately equalize signal intensities in the two channels. Image processing and data extraction were performed using GenePix Pro 3.0.6 (Axon Instruments). Data Analysis: Average background signal from negative control spots was subtracted from Cy3 and Cy5 signal intensities. In cases where background was larger than signal, signal was set to 1. Log ratios were calculated for each spot and log ratios from replicate spots were averaged. Data was normalized using the “Lowess” method, and analyzed for differential expression. Differentially expressed genes were defined as those with log transformed expression ratios more than 1.5 SD from the mean. This lower threshold was chosen since comparison of the microarray dat to qRT-PCR on the samples used for the microarray experiment indicated that the data was compressed. For example, TDGF1 was up-regulated 100-fold in uES versus EB by qRT-PCR, but only ~5-fold by microarray analysis in the same sample. Normalization and identification of differentially expressed genes was performed using the MIDAS package (Saeed et al. (2003) Biotechniques 34(2):374-8). Keywords: other

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The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters

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