tissue: Whole blood subject 1: Sample-104 subject 1 gender: Male subject 1 age: 39 years subject 2: Sample-112 subject 2 gender: Male subject 2 age: 60 years
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified from 200 µL of serum samples, using the PureLink Viral RNA/DNA Mini Kit (Invitrogen, USA), following manufacturer’s recommendations.
Label
Cy3
Label protocol
Individual or pool of equimolar amount of cDNAs were used for a random PCR amplification, which contained 1 nmol of the fluorescent labelling Cy3-dUTP (SIGMA ALDRICH, USA).
Hybridization protocol
PCR products were hybridized against the DNA microarray using the Oligo aCGH/ChIP-on-chip Hybridization Kit (Agilent, USA), following manufacturer’s recommendations.
Scan protocol
The slides were scanned with the Axon GenePix 4000B scanner (Molecular Devices, USA) at a 532 nm wavelength with a pixel size of 10-μm resolution.
Description
256203910011_8-8
Data processing
The median fluorescence intensity of each spot with local background subtraction was calculated from the scanned images using GenePix Pro 7 software (Molecular Devices, USA). The raw data of the sports were normalized against the negative control probes, which were randomly distributed on each sub-array. The normalized data were log2-transformed to reduce variability. The spots containing the probes for each virus species were grouped, and the mean signal intensity of all groups was calculated. The mean signal intensity of the group of probes corresponding to each virus species was compared to the mean signal intensity of the negative control probes, using the Welch’s t-test. A virus was considered present in an analyzed sample when the mean signal intensity of the group of probes was significantly (p<0.05) higher than the mean signal intensity of the negative control probes and showed a normalized mean intensity of at least 2, i.e., it was at least fourfold higher than the mean signal intensity of the negative control probes, thus reducing the chance of virus misidentification due to cross-hybridization.
