Diffusion, mixing, and associated dye effects in DNA-microarray hybridizations.
ABSTRACT: Typical DNA microarrays utilize diffusion of dye-labeled cDNA probes followed by sequence-specific hybridization to immobilized targets. Here we experimentally estimated the distance typical probes travel during static 16-h hybridizations. Probes labeled with Cy3 and Cy5 were individually introduced to opposite sides of a microarray with minimal convective mixing. Oppositely labeled probes diffused across the initial front separating the two solutions, generating a zone with both dyes present. Diffusion-distance estimates for Cy3- and Cy5-labeled cDNAs were 3.8 mm and 2.6 mm, respectively, despite having almost identical molecular masses. In separate 16-h hybridization experiments with oppositely labeled probes premixed, arrays that were continuously mixed had 15-20% higher signal intensities than arrays hybridized statically. However, no change was observed in the Cy3/Cy5 signal intensity ratio between continuously mixed and static hybridizations. This suggests that the observed dye bias in diffusion-distance estimates results from differences in the detection limits of Cy3 and Cy5-labeled cDNA, a potential concern for array data on low-abundance transcripts. Our conservative diffusion-distance estimates indicate that replicate targets >7.6 mm apart will not compete for scarce probes. Also, raising the microarray gap height would delay the onset of diffusion-limited hybridization by increasing the amount of available probe.
Project description:Vascular Endothelial Growth Factor (VEGF) is a critical regulator of pulmonary Th2 inflammation but the underlying mechanism and the roles of miRNAs in this process have not been defined. We analyzed the effect of VEGF on lung microRNAs by microarray analysis and validated the findings by Taqman qRT-PCR. We compared the levels of microRNAs in the lungs of transgenic mice with their wild type litters after overexpression of VEGF from a lung epithelium-restricted transgene. VEGF was overexpressed in the lung epithelium of CC10-rtTA-VEGF transgenic mice by adding Doxycycline to their drinking water for 7-10 days. RNA from the lungs of VEGF transgenic mice and their wild type litters were extracted and analyzed by microarray. The expression levels of microRNAs that were significanly altered were validated in another group of mice. Lung RNA from each transgenic mouse was labeled with Cy5 and lung RNA from wild type mice were labeled with Cy3. The two labeled RNAs were hybridized to the miRNA array. This array (chip ID 01_M9.1_070362) detects miRNA transcripts listed in Sanger miRBase Release 9.1 (http://www.sanger.ac.uk/Software/Rfam/mirna/). Multiple control probes are included in each chip. The control probes are used for quality controls of chip production, sample labeling and assay conditions. Among the control probes, PUC2PM-20B and PUC2MM-20B are the perfect match and single-based match detection probes, respectively, of a 20-mer RNA positive control sequence that is spiked into the RNA samples before labeling. One may assess assay stringency from the intensity ratio of PUC2PM-20B and PUC2MM-20B, which is normally larger than 30. When the option for custom probes is selected, custom probes are also included. After hybridization three imaged were obtained. From Cy3 and Cy5 images one may directly read miRNA profiles and from Ratio images one may get a quick sense of differential expressions between the corresponding samples. The images are displayed in pseudo colors so as to expand visual dynamic range. In the Cy3 and Cy5 intensity images, as signal intensity increases from 1 to 65,535 the corresponding color changes from blue to green, to yellow, and to red. In the Cy3/Cy5 ratio image, when Cy3 level is higher than Cy5 level the color is green; when Cy3 level is equal to Cy5 level the color is yellow; and when Cy5 level is higher than Cy3 level the color is red.
Project description:RNA was extracted with Rneasy Micro Kit (Qiagen, France) from SKBR3 cells treated with 1 uM ATRA. The quantity and purity of the extracted RNA was evaluated using a NanoDropTM spectrophotometer and its integrity measured using an Agilent BioanalyzerTM. For microarray hybridizations, 500 ng of total RNA from each RNA sample was amplified and labeled with two fluorescents dye (Cy5 and Cy3) using the Quick Amplification Labeling kit (Agilent Technologies, Palo Alto, CA, USA) following the manufacturer's protocol. Cy3-labeled and Cy5-labelled cRNA were hybridized to the Agilent Human Whole Genome Oligo Microarray format 4x44K (Agilent Technologies), prior to washing and scanning.
Project description:Gene expression analysis was conducted using Agilent Human1Av2 arrays (Agilent Technologies, Palo Alto, CA). Two separate biological replicates of cytoplasmic RNA samples were harvested and purified from each of the U-2 OS cell lines stably expressing hGRalpha, -A, -B, -C3, or D3 treated with 100 nM DEX or vehicle for 6 hours using RNeasy Midi kits (Invitrogen). These RNA samples were amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 0.5 ng of total RNA, two cRNAs, one labeled with Cy3 and one labeled with Cy5 were produced for each sample according to the manufacturerâ??s protocol. Consequently, four separate chips were used for each of the isoforms at each treatment condition, yielding a total of 40 chips: 5 isoforms X 2 treatments (vehicle vs. hormone) X 2 biological replicates X 2 labeling dyes. For each two-color comparison, 750 ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 16 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data were obtained using the Agilent Feature Extraction software (v7.1), using defaults for all parameters.
Project description:Assays 1-4: Effects of DnaA activity on S. meliloti chromosome, pSymA and pSymB replicon bias. Sheared DNA extracts of IPTG-induced vs uninduced cultures of DnaA (Assays 1,2) or Hda (Assays 3,4) overexpressors were labeled with Cy3 or Cy5 and hybridized applying dye-swap replicate design. Assay 5: Hybridization of sheared DNA of logarithmic (Cy3-labeled) vs stationary phase (Cy5-labeled) culture.
Project description:A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding approximately 500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods.
Project description:We used the commercially available amino-allyl RNA amplification Kit ver,2 (High Yield Type) (SIGMA-ALDRICH). Purified total RNA (3 ?g) was reverse-transcribed to generate double-stranded cDNA using an oligo dT T7 promoter primer and reverse transcriptase. Next, cRNA was synthesized using T7 RNA polymerase, which simultaneously incorporated Cy3- or Cy5-labeled cytidine triphosphate. During this process, the samples of SP cells were labeled with Cy5, whereas the non-SP cells were labeled with Cy3 as control cells. Quality of the cRNA was again checked using the Nano Drop. Cy3-labeled cRNA and Cy5-labeled cRNA were combined and then fragmented in a hybridization cocktail (SIGMA-ALDRICH). Then the labeled cRNAs were hybridized to a 60-mer probe oligonucleotide microarray and incubated for 20 h ours at 50 ?C. The fluorescent intensities were determined by a Genepix 4000B Microarray Scanner (Axon, US).
Project description:This SuperSeries is composed of the following subset Series:<br><br> GSE3838: Temporal expression profile of CHRF-288 cell line after phorbol ester stimulation <br>CHRF-288 cells were cultured in the presence of 10 ng/mL phorbol ester (PMA) or equivalent volume of DMSO solvent (0.02%). Unstimulated control cells from time zero (exponentially growing CHRF cells) were also analyzed. For PMA treated legs, only the adherent cells were included in the transcriptional analysis. Two biological replicate experiments were analyzed and approximately one-half of the samples with each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference RNA pool labeled with Cy5.<br><br> GSE3839: Temporal expression profile of megakaryocytic differentiation primary CD34+ cell culture Experiment <br> G-CSF mobilized peripheral blood CD34-positive cells were cultured with TPO, IL-3, and Flt3-L to induce Mk differentiation. Samples prior to day 5, including uncultured starting cells, were analyzed directly, whereas samples after and including day 5 were positively selected for CD41a expression immediately prior to RNA isolation. Three biological replicate experiments were analyzed and approximately one-half of the samples from each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference RNA pool labeled with Cy5.
Project description:BACKGROUND: RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array) procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. RESULTS: RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. CONCLUSION: RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.
Project description:BACKGROUND: Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard. RESULTS: We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable. CONCLUSION: RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.
Project description:In order to assess global differences in gene expression that may be regulated directly or indirectly by HPr , a global transcriptomic profile analysis of the Î?ptsH mutant and the wild-type MC58 strain was performed. cDNA probes were prepared from RNA pools (5 Î¼g) obtained from MC58 wild-type and Î?ptsH mutant cells grown in rich GCB broth until mid-logarithmic phase. RNA was extracted from mid-log phase and differential gene expression through microarray analysis was performed. Equal amounts of Cy5- and Cy3-labelled cDNAs were hybridized onto the microarray for 17 h at 65Â°C according to the Agilent protocol. Three hybridizations were performed using cDNA probes from three independent pools, respectively, corresponding to RNA extracted from a total of nine independent bacterial cultures for each strain. Images were acquired by using an Agilent microarray scanner G2505B at 5 Âµm of resolution and using the extended dynamic range and were analysed with Agilent Feature Extraction 9.5.1. Differentially expressed genes were assessed by grouping all log2 ratios of the Cy5 and Cy3 values corresponding to each gene, within experimental replicas and spot replicas, and comparing them against the zero value by Student's t test statistics.