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FAQ

Spotted Arrays:

After you get your results:

Spotted Microarrays:

What is a spotted DNA microarray?

Answer: In this approach, distinct DNA fragments (cDNA or oligonucleotides) are attached as an array of distinct spots on a suitably treated glass microscope slide via a mechanical robotic spotting process. Two distinct probe DNA or mRNA mixtures - the reference and the test sample - are given fluorescent red and green labels and are combined in solution and applied to the array. The relative amounts of red and green fluorescence at each spot provide a measurement of the relative numbers of red and green labeled fragments attached at the spot, and thus of the relative numbers of fragments in the reference and test samples. The two-color system requires a compatible pair of dyes. The most commonly used are the Cy5 (Red) and Cy3 (green) fluorescent dyes. These dyes are relatively bright, stable, and they fluoresce when dry, so that the hybridized arrays can be fluorescently imaged in a dry state. On the other hand, these are patented, proprietary dyes, and their cost accounts for more than half of the cost of a spotted microarray experiment.

The probe typically consists of red fluorescently labeled mRNA (or, more commonly, the corresponding cDNA produced by in vitro reverse transcription) extracted from a test sample, and a green fluorescently labeled cDNA from a reference sample. These labeled probes are mixed in solution and hybridized to the array. Unbound probe is washed away, and the result is scanned by a fluorescent imaging system to yield red (upregulated), green (downregulated) and yellow (no difference) intensity measurements from each spot on the array. The ratio of these red and green intensities - suitably normalized - provides a measure of the change in mRNA levels between the test and reference populations, and thus of relative levels of gene expression in the test and reference sample. Compared to traditional techniques, this procedure is analogous to simultaneously carrying out thousands of Northern blots. Type of sample comparisons and experimental design used in for data analysis include direct and indirect comparisons, these are briefly mentioned below.

i . Direct comparisons include the basic comparative measurement for comparison of two samples. A reference sample is tissue or a cell line that is an "normal" state, and the test sample if from the same type of cells in a diseased or otherwise altered state. In this case, the red/green ratio provides a direct comparison of the expression in the normal and altered cells for each gene on the array.

ii. Another type of comparison includes the indirect sample comparison. To make all direct comparisons between N samples requires on the order of NxN comparisons, which gets large quickly. Instead, to compare or characterize many test samples, it is convenient to compare each one to a universal reference sample. This can be any mix of DNAs that reliably and repeatably light up most spots on the array (so that the red/green ratio is meaningful at each spot), and is available in large enough quantities to use the same batch for many experiments. Convenient reference samples can be made for mixes of RNAs from several specific cell lines or tissues. In this approach, all test samples are hybridized to the reference sample, and compared to each other only indirectly using the results of their experimental comparisons to the reference.

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What spotted microarrays are available in the facility? Can I get a genelist for available arrays?

Answer: Please to go our Spotted Array Page, find the table of available arrays. You can download the genelist for all available arrays there.

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What's the price of spotted microarrays?

Answer: Please go to our Spotted Array Page, which has a table of prices for all the services we offer.

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How much RNA is needed to run a spotted microarray?

Answer: 10μg for total RNA in 10μl RNase-free water. We also need to do an Agilent RNA quality check before labeling and hyb, so please also provide 3μl of the same sample at the same concentration in a seperate tube. If you do not have enough RNA, you may have to amplify your samples before labeling. Please click here for these requirements.

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What should I put in the submission form for Cy5 and Cy3 samples?

Answer: Generally you'll put your samples on one channel (usually Cy5) and put reference RNA on the other channel (usually Cy3). The relative intensity signals are presented as a ratio Cy5/Cy3 in the result file. For Human, Mouse and Rat we have universal reference RNA from Stratagene. For other organisms you have to provide your own reference RNA. For microRNA assays we recommend using Ambion's synthetic miRNA Reference Panel.

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After you get your results

What options do I have for data analysis after I receive the results?

Answer: If you choose to do your own analysis, we can help you with any supported software listed on our Data Analysis Page.

We can also perform data analysis for you. Click here for more information.

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What are those numbers in the summary file I received with my spotted array results?

scan date: the date your arrays were scanned.

Genome ID: the system we use to track individual hybridizations. Every sample should have a distinct genome ID.

Array: the array you are using. e.g. MO30K means it's Mouse array; the oglios are from Operon; the size is 30K genes.

Sample: sample name. Cy5 sample name vs Cy3 sample name

635 Signal/Background: the signal to background ratio for the Cy5/635 channel. should expect this number to be above 2.

532 signal/background: the signal to background ratio for the Cy3/532 channel. should expect this number to be above 2.

635 background: the average (mean of median) background intensity for Cy5 channel, should expect this number to below 200, most time below 100.

532 background: the average (mean of median) background intensity for Cy3 channel, should expect this number to below 200, most time below 100.

635 signal/noise: the median of signal to noise ratio for Cy5 channel, should expect this number to above 2, higher than 5 means the Cy5 sample hybridized very well.

532 signal/noise: the median of signal to noise ratio for Cy3 channel, should expect this number to above 2, higher than 5 means the Cy3 sample hybridized very well.

PMT 635: the laser power we used to scan the Cy5 channel, usually 400-700.

PMT 532: the laser power we used to scan the Cy3 channel, usually 400-700.

F635 Median: the median foreground intensity for Cy5 channel, usually 300 and up.

F532 Median: the median foreground intensity for Cy3 channel, usually 300 and up.

not found: the percentage of not found features -- the percentage of spots didn't get hybridized. Depend on samples you have and which array you are working on. If it's below 25%, means alomst all the genes on that array were detected in your samples (can be from either the Cy5 sample or Cy3 sample, or both.)

Cy5 RNA concentration: the start Cy5 sample concentration we tested on Nanodrop.

Cy3 RNA concentration: the start Cy3 sample concentration we tested on Nanodrop.

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What are the result files for a spotted array? What does each column in a .gpr file mean?

Answer: Data acquisition is performed using the Axon GenePix Pro 4000A interface. Axon GenePix scanner refers to spots on an array as a feature. There are two dyes used in the hybridization, Cy5 and Cy3. Cy5 is scanned at a wavelength of 635 and fluoresces the color red. Cy3 is scanned at wavelength 532 and fluoresces green. The Axon GenePix software refers to the intensities of the spots by the wavelength at which they are scanned. The Duke Microarray Facility calculates all ratios as intensity of Cy5 signal (red) / Intensity of signal Cy3 (green). If a ratio is equal to 2, then the expression of the feature was twice as high in the Cy5 labeled sample when compared to the Cy3 labeled sample. The same type of logic holds true when the intensity of the Cy3 signal is greater than the intensity of the Cy5. The ratio would be represented as a number less than 1.

For each array experiment that is completed, five files are generated. Each file will be named identical except for the extension. The naming convention is as follows:

Project ID number_Genome ID number_slidenumber_slidelot_chip type_Cy5sample_Cy3sample (for example, 0089_477_001_01_HO21K_ko_wt.*)

Extension Description Comments
*.TIFF (635nm and 532nm) Image file, picture of scanned array Can be viewed in Photoshop and Powerpoint
*.GPR Tab- deliminated text file with the raw data, see table below for content. Can be opened in Excel to manipulate
*.JPG Shows the array image with both channels overlaid Can open in any operating system
*.GPS Gene Pix Settings File, acquition, analysis and display settings are saved as binary GenePix settings files. Settings are organized into several different categories (acquisition, display, and analysis) all of which are saved together in the GPS file. This file contains block and feature geometry, and can be used to apply a grid template to an image. Used with the Axon GenePix software

The data analysis out put file is the *.GPR file. A description of each column in the GPR file are listed below:(info from http://www.axon.com/gn_GPR_Format_History.html)

Column Title Description
Block the block number of the feature.
Column the column number of the feature.
Row the row number of the feature.
Name the name of the feature derived from the Array List (up to 40 characters long, contained in quotation marks).
ID the unique identifier of the feature derived from the Array List (up to 40 characters long, contained in quotation marks).
X the X-coordinate in m of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image.
Y the Y-coordinate in m of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image.
Dia the diameter in m of the feature-indicator.
F635 Median median feature pixel intensity at wavelength #1 (635 nm).
F635 Mean mean feature pixel intensity at wavelength #1 (635 nm).
F635 SD the standard deviation of the feature pixel intensity at wavelength #1 (635 nm).
F635 CV the coefficient of variation of feature pixel intensity.
B635 the actual background value used for the feature in GenePix Pro calculations (as opposed to B635 Median, for example, which is the local median background.) This column is required because GenePix Pro 5.0 has global and negative control background subtraction methods. If you choose a non-local method, B635 is different to B635 Median
B635 Median the median feature background intensity at wavelength #1 (635 nm).
B635 Mean the mean feature background intensity at wavelength #1 (635 nm).
B635 SD the standard deviation of the feature background intensity at wavelength #1 (635 nm).
B635 CV the coefficient of variation of background pixel intensity.
% > B635 + 1 SD the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #1 (635nm).
% > B635 + 2 SD the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #1 (635nm).
F635 % Sat. the percentage of feature pixels at wavelength #1 that are saturated.
F532 Median median feature pixel intensity at wavelength #2 (532nm).
F532 Mean mean feature pixel intensity at wavelength #2 (532nm).
F532 SD the standard deviation of the feature intensity at wavelength #2 (532nm).
F532 CV the coefficient of variation of feature pixel intensity.
B532 the actual background value used for the feature in GenePix Pro calculations (as opposed to B532 Median, for example, which is the local median background.) This column is required because GenePix Pro 5.0 has global and negative control background subtraction methods. If you choose a non-local method, B532 is different to B532 Median
B532 Median the median feature background intensity at wavelength #2 (532nm).
B532 Mean the mean feature background intensity at wavelength #2 (532nm).
B532 SD the standard deviation of the feature background intensity at wavelength #2 (532nm).
B532 CV the coefficient of variation of background pixel intensity.
% > B532 + 1 SD % > B532 + 2 SD
F532 % Sat. the percentage of feature pixels at wavelength #2 that are saturated.
Ratio of Medians the ratio of the median intensities of each feature for each wavelength, with the median background subtracted.
Ratio of Means the ratio of the arithmetic mean intensities of each feature for each wavelength, with the median background subtracted.
Median of Ratios the median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted.
Mean of Ratios the arithmetic mean of the pixel-by-pixel ratios of pixel intensities, with the median background subtracted.
Ratios SD the standard deviation of pixel intensity ratios.
Rgn Ratio the regression ratio.
Rgn R2 the coefficient of determination for the current regression value.
F Pixels the total number of feature pixels.
B Pixels the total number of background pixels.
Circularity a measure of circularity from 0 to 100, using a metric based on the variance of the distance of each boundary pixel to the centroid of the feature: 100 is most circular, 0 is most non-circular. Circular features always have a circularity of 100, square features always have a circularity of 79 (= p/4*100).
Sum of Medians the sum of the median intensities for each wavelength, with the median background subtracted.
Sum of Means the sum of the arithmetic mean intensities for each wavelength, with the median background subtracted.
Log Ratio log (base 2) transform of the ratio of the medians.
F635 Median - B635 the median feature pixel intensity at wavelength #1 with the median background subtracted.
F532 Median - B532 the median feature pixel intensity at wavelength #2 with the median background subtracted.
F635 Mean - B635 the mean feature pixel intensity at wavelength #1 with the median background subtracted.
F532 Mean - B532 the mean feature pixel intensity at wavelength #2 with the median background subtracted.
F635 Total Intensity the sum of all pixel intensities in the feature.
F532 Total Intensity the sum of all pixel intensities in the feature.
SNR 635 the signal-to-noise ratio of the feature, calculated as (F635 Mean - B635 Mean) / B635 SD.
SNR 532 the signal-to-noise ratio of the feature, calculated as (F635 Mean - B635 Mean) / B635 SD.
Flags the type of flag associated with a feature.
Normalize flag column describing if the feature was used to calculate the normalization factors (1 for used, 0 for not used).
Autoflag reports whether or not a feature has been flagged from the Flag Features dialog box. It applies to good and bad flags only.

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I'm using GeneSpring. Where can I get the genome file for spotted arrays?

Answer: Please go to our Spotted Array Page, where you can download GeneSpring genome files.

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