One-half µg RNA of each sample was amplified by Illumina TotalPrep RNA Amplification Kit according to the manufacturer’s instructions (Ambion, Austin, TX).
Label
Cy3
Label protocol
After non-specific binding targets were washed, the hybridization arrays were conjugated with fluorescent detector of Strepavidin-Cy3.
Hybridization protocol
Then, 10 µg of fragmented biotin-labeled cRNA was hybridized on Phalanx Human OneArray™ by Phalanx hybridization buffer at 50oC in oven for 14-16 hrs using the bubble-mixing method.
Scan protocol
Finally, arrays were dried by centrifugation and scanned by DNA Microarray Scanner (Agilent Technologies, Santa Clara, US). Images from the scanned arrays were quantified using GenePix® Pro 4.0 (Molecular Devices, Sunnyvale, CA).
Description
sample 106
Data processing
Spots in each array with foreground median intensity of wavelength 532 nm greater than or equal to that of background median intensity plus 3 folds standard deviation of wavelength 532 nm were considered as the “Present” flag and included for the further analysis. In order to evaluate the quality of each array in the entire array experiment, three evaluation steps were performed: basic, reproducible, and diagram. In basic step, three parameters, including percentage of “Present” spots among all spots, the average intensity of “Present” spots, and coefficient of variation of intensity for control spots in the entire arrays were all considered. If any two parameters in one array were located outside the 1.5-folds interquartile range (25th-75th) of same parameters for all arrays, that array was excluded. The remaining arrays were then evaluated in reproducible steps which the repeated arrays of the same sample would pass, when their Pearson’s correlation coefficient was larger than 0.95 and “2-fold percentage” was less than 15% (sFig. 4). The “2-fold percentage” was the percentage of probes among all probes in which the ratio of the same probe between two arrays exceeded 2-fold. In the final diagram step, the density plot of repeated arrays was used to examine the intensity profile of each array. An array would pass if the profile was similar to the rest of arrays in the same phenotype groups. When the arrays passed all three steps, the raw intensity of spots were log-2 transformed for subsequent analysis. To adjust the systematic variation of experiments and dye effects, global Lowess normalizations were performed within repeated arrays of the same sample and between the samples. Spot was included for further analysis when it was “Present” in at least one of the qualified arrays.
