NIA Array Analysis Tool

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, universal reference(UR))

Geneid	Brain-rep1	UR-rep1	Brain-rep2	UR-rep2	Brain-rep3	UR-rep3	Liver-rep1	UR-rep1	Liver-rep2	UR-rep2	Liver-rep3	UR-rep3	Ovary-rep1	UR-rep1	Ovary-rep2	UR-rep2	Ovary-rep3	UR-rep3
1	397	566	361	428	290	460	444	387	580	524	509	403	540	481	471	410	464	477
1	17588	24026	21732	28250	14945	19010	42984	19784	41190	19296	15322	23817	15305	23460	39107	32317	34062	20783
1	13885	19448	14564	18401	10715	18316	22732	13898	22419	18699	20509	13856	22930	19980	27063	20288	17876	17878
1	185	198	124	110	114	94	47	83	140	97	118	88	96	104	110	87	102	109
1	8050	5973	6297	5745	6180	5558	5208	4988	6691	6506	5057	5021	5846	5634	6308	6405	5357	5072
1	9876	23492	15672	24722	14338	22847	17898	19842	20443	24817	10827	19323	12557	25309	19961	28777	14401	22616
1	2566	1438	3155	1447	3129	1353	3661	1145	4442	1436	3909	1181	5066	1473	4391	1630	3462	1287

To upload the file, click the "Browse..." button near field "Upload new data file". Do NOT use normalization or split-normalization. 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 "2-color arrays".

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

Because UR is used only as a reference, check that the field "don't analyze reference" is selected. You can put "UR" into the Reference name field.

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, then the input file would have 36 data columns instead of 18. Then the lower table will have numbers: 2 2 2 2 2 2 2 2 2. There are 3 experiments, each replicated 3 times, so there are 9 replications total, and for each we specify the number of subreplications.