Comparison of Hybridization and Wash Procedures for Agilent 60mer Oligonucleotide Microarrays
Jenny XiaoAnne Bergstrom LucasGlenda DelenstarrRhoda Argonza-BarrettErik BjeldanesLou WelebobKaho MinouraPaul K. Wolber
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Objective To optimize the preparation and hybridization conditions of long oligonucleotide microarray for Astrovirus detection.Methods To optimize the probe concentration spotted on the slide,UV cross-linking energy,the hybridization time and temperature by dye primer.Results The stable hybridization signal was obtained at the probe spotting concentration of 10 μmol/L.On the condition cross-linking at 3 600 mJ we got best immobilization results.The optimized hybridization buffer is composed of 50 % formamide,5×SSC,0.5 %SDS and 1% salmon sperm DNA.The optimal hybridization time and temperature was 8 hours and 65 ℃ respectively.Conclusion The preparation and hybridization conditions of 70 mer oligonucleotide microarray was eptimized.This study may lay the foundation for the parallel detection of different species of viruses in marine environment.
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The ability to measure steady-state mRNA levels is central to the analysis of neuronal gene expression and, therefore, finds application across a broad range of neuroscientific research endeavors including the investigation of spatial-, temporal-, drug-induced-, or activity-dependent-differential gene expression, The actual measure required, however, depends to a large extent upon the experimental paradigm. For example, one of the first questions often addressed following the cloning of new genes is that of the tissue distribution of its expression, for which the technique of northern blotting (Alwine et al., 1977) is widely used (for example, see Webb et al., 1993). More detailed analyses of spatial expression patterns can be performed using in situ hybridization histochemistry (for example see Glencorse et al., 1993; Webb et al., 1994), the subject of a separate review in this volume (Bateson, 1998). The study of differential gene expression through development has also predominantly relied upon the techniques of Northern blotting and in situ hybridization histochemistry (for example see MacLennan et al., 1991; Laurie et al., 1992). These two methodologies are relatively straightforward to perform and allow the investigator to readily identify a brain region and/or cell type, or a developmental time point, where the gene of interest displays "maximal" expression without the need for rigorous quantification of mRNA levels.
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Objective To study the effect of oligonucleotide probe length on hybridization signals in order to fabricate cost-effective expression profiling microarrays. Methods The effect of oligonucleotide probe length on hybridization signals was studied through expression profiling of E.coli. Twenty E.coli genes,which represent low,median and high expression level respectively were chosen from the E.coli microarray database.Oligo probes of 59-mer and 70-mer,the former being more cost-effective than the latter,were designed and printed on a same slide.Positive control and spotting buffer as negative control were also spotted on the slide.Fluorescence labeled DNA were prepared from total RNA of E.coli through reverse transcription and RNA amplification,then hybridized with the array.After hybridization,slides were scanned with a confocal scanner and hybridization signals were extracted from images.Results The quality of total RNA was assessed,showing the 28S rRNA and 18S rRNA was intact.Our results indicated that there were no significant differences between the 59-mer probes and 70-mer probes in the hybridization efficiency and signal intensity(P= 0.9810).In addition,the positive probes had strong intensity signal,while the negative spots had no signal as expected. Conclusions The cost-effective 59-mer oligo probes could be used to fabricate the expression profling microarray while retain a comparable result with longer probes.This can help to promote the extensive application of DNA microarray technology.
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We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.
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