NIA Array Analysis Tool



Comparing multiple tissues with 2-color arrays

We assume that comparison of multiple tissues with 2-color arrays implies the use of Universal Reference in the second color channel. You can use any other reference instead of the universal reference. For example, control or the zero-point in a time-series can be used as a reference. The important thing is that the same reference should be used in all arrays.

The input file should be a tab-delimited text the specified format. If you use log-transformed data check the corresponding box. Data from 2 color channels should be in adjacent columns. You don't need to use dye-swap with universal reference reference. However if your reference is important for mean comparison (e.g., control or zero-point in a time-series), then you need dye swap; if you use dye swap, then data columns should be swapped. Replications are grouped together, as shown in the example below.

Example of an input file (3 tissues, 2-color arrays)

Geneid
Brain-rep1
Brain-rep2
Brain-rep3
Liver-rep1
Liver-rep2
Liver-rep3
Ovary-rep1
Ovary-rep2
Ovary-rep3
1
180
314
296
433
182
311
397
566
361
2
6780
17085
9223
18468
8623
15019
17588
24026
21732
3
16592
15161
10476
16790
9752
10316
13885
19448
14564
4
2896
239
101
59
29
53
185
198
124
5
5496
4283
6635
4912
4459
3175
8050
5973
6297

Or a simpler way:
Geneid
Brain
Brain
Brain
Liver
Liver
Liver
Ovary
Ovary
Ovary
1
180
314
296
433
182
311
397
566
361
2
6780
17085
9223
18468
8623
15019
17588
24026
21732
3
16592
15161
10476
16790
9752
10316
13885
19448
14564
4
2896
239
101
59
29
53
185
198
124
5
5496
4283
6635
4912
4459
3175
8050
5973
6297

You can always change tissue names after the file was loaded, so you don't need to edit column headers. However, if column headers are correct then you can use the option (checkbox) to use column headers, and then you will not need to edit them after loading.

To upload the file, click the "Browse..." button near field "Upload new data file". If you need normalization, check the "normalize" box. If you need split-normalization, use 2nd column of the input file for array index and check the "split-normalize" box. Then push "Upload" button. After the file is loaded push "Continue". After the file is downloaded, some default parameters are already filled in. However you need to modify some of them to specify your experiment.

Description. Write a short description of your experiment, e.g., "Brain, Liver, Ovary".

Array type. Select the array type that you used. If the array type is not specified then you will not get references to gene symbols and annotations from graphs and tables. However you can upload you own array annotation if you click on the "Add array" button. See array annotation

. Number of colors in the array. Set it to "1-color arrays".

For this example there are 3 experiments (3 tissues), with 3 replications in each. Thus the table of experiments should look like this:

Other parameters can be left with their default values. However in some special cases you may need to change some of these parameters. Then use links from parameter names as a guide for changing.
WARNING: Do NOT change parameters if you don't understand their effect on program performance. First read the description of the parameter!

If you use subreplications, then fill out the bottom table. Otherwise leave it blank. For example, if each replication had 2 technical subreplications (i.e., each biological sample was hybridized twice), then the input file would have 18 data columns instead of 9. Then the lower table will have numbers: 2 2 2 2 2 2 2 2 2. There are 3 tissues, each replicated 3 times, so there are 9 replications total, and for each we specify the number of subreplications.