Search

Background DNA microarrays, which have been increasingly used to monitor mRNA transcripts at a global level, can provide detailed insight into cellular processes involved in response to drugs and toxins. This is leading to new understandings of signaling networks that operate in the cell, and the molecular basis of diseases. Custom printed oligonucleotide arrays have proven to be an effective way to facilitate the applications of DNA microarray technology. A successful microarray experiment, however, involves many steps: well-designed oligonucleotide probes, printing, RNA extraction and labeling, hybridization, and imaging. Optimization is essential to generate reliable microarray data. Results Hybridization and washing steps are crucial for a successful microarray experiment. By following the hybridization and washing conditions recommended by an oligonucleotide provider, it was found that the expression ratios were compressed greater than expected and data analysis revealed a high degree of non-specific binding. A series of experiments was conducted using rat mixed tissue RNA reference material (MTRRM) and other RNA samples to optimize the hybridization and washing conditions. The optimized hybridization and washing conditions greatly reduced the non-specific binding and improved the accuracy of spot intensity measurements. Conclusion The results from the optimized hybridization and washing conditions greatly improved the reproducibility and accuracy of expression ratios. These experiments also suggested the importance of probe designs using better bioinformatics approaches and the need for common reference RNA samples for platform performance evaluation in order to fulfill the potential of DNA microarray technology.

Several Salmonella enterica serotypes were isolated from unpasteurized orange juice samples analysed as a follow-up to an outbreak in 1999 of S. enterica serotype Muenchen in the Pacific Northwest regions of United States. Eleven S. enterica strains were serotyped and identified as S. enterica serotype Muenchen (2), S. enterica serotype Hidalgo (2), S. enterica serotype Alamo (1), S. enterica serotype Gaminera (2), S. enterica serotype Javiana (2) and a new serotyped strain S. enterica serotype Tempe (2). The identity of the new serotype S. enterica serovar Tempe serotype 30:b:1,7:z33 was confirmed by the National Salmonella Reference Laboratory at NCID/CDC, Atlanta. These strains were sensitive to ampicillin, chloramphenicol, kanamycin, tetracycline, streptomycin and sulfisoxazole antibiotics. Isolates were screened for invasion (invA) and virulence (spvC) genes using specific primers for these two genes by polymerase chain reaction. All strains were positive for invA gene giving 321-bp fragment, however negative to virulence spvC gene. For pulsed-field gel electrophoresis (PFGE) analysis, Salmonella strain plugs were made and digested with XbaI and subjected to 18-h electrophoresis. The PFGE patterns were different for each S. enterica serotypes suggesting the several origins of contamination in outbreak. S. enterica serotype.

The efficacy of cleaning and sanitation in a small apple cider processing plant was evaluated by surface swab methods as well as microbiological examination of incoming raw ingredients and of the final product. Surface swabs revealed that hard-to-clean areas such as apple mills or tubing for pomace and juice transfer may continue to harbor contaminants even after cleaning and sanitation. Use of poor quality ingredients and poor sanitation led to an increase of approximately 2 logs in aerobic plate counts of the final product. Reuse of uncleaned press cloths contributed to increased microbiological counts in the finished juice. Finally, using apples inoculated with Escherichia coli K-12 in the plant resulted in an established population within the plant that was not removed during normal cleaning and sanitation. The data presented in this study suggest that current sanitary practices within a typical small cider facility are insufficient to remove potential pathogens.

Environmental ozone can rapidly degrade cyanine 5 (Cy5), a fluorescent dye commonly used in microarray gene expression studies. Cyanine 3 (Cy3) is much less affected by atmospheric ozone. Degradation of the Cy5 signal relative to the Cy3 signal in 2-color microarrays will adversely reduce the Cy5/Cy3 ratio resulting in unreliable microarray data. Ozone in central Arkansas typically ranges between ~22 ppb to ~46 ppb and can be as high as 60–100 ppb depending upon season, meteorological conditions, and time of day. These levels of ozone are common in many areas of the country during the summer. A carbon filter was installed in the laboratory air handling system to reduce ozone levels in the microarray laboratory. In addition, the airflow was balanced to prevent non-filtered air from entering the laboratory. These modifications reduced the ozone within the microarray laboratory to ~2–4 ppb. Data presented here document reductions in Cy5 signal on both in-house produced microarrays and commercial microarrays as a result of exposure to unfiltered air. Comparisons of identically hybridized microarrays exposed to either carbon-filtered or unfiltered air demonstrated the protective effect of carbon-filtration on microarray data as indicated by Cy5 and Cy3 intensities. LOWESS normalization of the data was not able to completely overcome the effect of ozone-induced reduction of Cy5 signal. Experiments were also conducted to examine the effects of high humidity on microarray quality. Modest, but significant, increases in Cy5 and Cy3 signal intensities were observed after 2 or 4 hours at 98–99% humidity compared to 42% humidity. Simple installation of carbon filters in the laboratory air handling system resulted in low and consistent ozone levels. This allowed the accurate determination of gene expression by microarray using Cy5 and Cy3 fluorescent dyes.