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Ocimum Hybridization Services offer an easy and affordable way towards good, reproducible microarray results.
According to your requirements you can choose from the following services:
Please Note:
The hybridisation service can be ordered with all OciChip™ arrays (catalog arrays and custom arrays). We run control samples with every hybridisation service. Should your samples show insufficient results while these controls perform normally, we will charge you a setup fee only.
Ocimum Labeling
Depending on the amount of total RNA and your requests, we will perform direct incorporation (reverse transcription of RNA into cDNA, incorporating Cy3 (Cy5)-dCTP), Micro Labeling Protocol (hybridization with amplified cRNA, incorporating Cy3/Cy5 via aminoallyl) or our Nano Labeling Protocol (hybridization with 2x amplified cRNA, incorporating Cy3/Cy5 via aminoallyl).
Ocimum Hybridization
According to your request, we perform single-dye hybridizations or co-hybridizations (two differentially labeled samples per slide). Hybridization and stringency washes are performed at conditions that assure highest possible signal intensity and uniformity, at lowest background contamination. We use salt-based hybridization buffers in a highly automated procedure. Labeled samples are concentration-adjusted in relation to yield and incorporation rate for improved performance.
Ocimum Scanning
Every channel (Cy3, Cy5) is scanned with a laser scanner (10 µm resolution) at three different photomultiplier (PMT) gain settings. The produced 16-bit TIFF images are used for analysis.
Ocimum Analysis
- Software tools Genowiz™ (Ocimum Biosolutions), and MAVI (Ocimum Biosolutions) are used to perform microarray data analysis.
- The scanned images are analyzed to digitalize spot signals. Unique pixel selection algorithms determine signal and background intensities for every individual spot.
- These results are further processed using MAVI. This proprietary software increases the dynamic range, while at the same time avoiding saturation problems. This is achieved by linear regression analyses on intensity data of images from three different scanner PMT gain settings.
- MAVI also calculates normalization parameters, using the average intensity of each channel or your selection of housekeeping genes. A log file displays quality parameters.
- MAVI result files are imported into individual Excel sheets for each slide, combining the array with gene description data. These Excel sheets contain all raw data, normalized and background corrected intensities as well as ratio values, calculated according to your preferences.
- Genowiz™ is used to visualize the data, creating histogram (ratios) and scatter plots (intensities).
- If dye swap experiments are performed, the average of both slides is calculated additionally.
Further analysis (clustering, statistics) is available on request.
Required specifications for your samples
Total RNA samples (from non-infectious sources only):- Have to be DNase I treated OD 260/230 >= 2, OD 260/280 >= 1.8
- For direct incorporation labeling > 100 µg total RNA, for Micro Labeling > 1 µg total RNA, and for Nano Labeling > 100 ng total RNA are required
- Adjust concentration to 6µg/µl (direct labeling) or 1 µg/µl (Micro Labeling ) in H2O; for Nano Labeling we recommend to send > 100 ng of total RNA ethanol precipitated with a stained carrier (e.g. "Pellet Paint")
- Please provide an image (formaldehyde-agarose (1.3%) gel electrophoresis) together with your total RNA samples
- Please ship your total RNA samples on dry ice
Cy3- and Cy5 -labeled samples:- Please provide OD 260/280, amount and incorporation rate, if possible Minimum 1 µg labeled cDNA or 10 µg labeled, fragmented cRNA are required
- Please dry your samples (speed-vac) and ship light protected
Hybridized arrays:- Please ship the slides protected from dust and light
Recommendations for experimental set-up
OciChip™ allows you to choose between single dye and co-hybridization experiments.
When performing co-hybridizations, we highly recommend performing reverse labeling (dye swap) of your samples. The additional slide needed will highly improve your data quality and avoid false positives. Single dye hybridizations increase the dynamic range of your differential expression data, but are more susceptible to background. With our experience in performing microarray experiments, background is usually not a problem. Depending on your needs you can therefore choose between both set-ups. We will gladly assist you in designing an experimental set-up that best fits your requirements.
Turn-around-time: Please allow 2 - 4 weeks
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OciChip™ array Concept
