Laboratory viruses were propagated in C6/36 or Vero cells using Leibovitz's L-15 medium containing 10% fetal bovine serum (FBS) (Cultilab, Brazil). The Zika virus was obtained from Baby mice cerebrum.
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified from 140 µL of the supernatant of infected and uninfected cell cultures with the AxyPrep viral RNA/DNA Miniprep Kit (Axigen, USA), following the manufacturer’s recommendation. The RNA was stored at -80ºC until use.
Label
Cy3
Label protocol
The synthesis of target cRNA probes and hybridization were performed using Agilent’s Low Input Quick Amp WT Labeling kit, one color (Agilent Technology, USA), according to the manufacturer’s instructions. Briefly, total RNA was mixed with the WT primer mix, a 1:5000 dilution of the positive RNA control (Agilent, USA) and incubated at 65 ºC for 10min. cDNA master mix (5X first strand buffer, 0.1M DTT, 10mM dNTP mix, RNase-Out, and MMLV-RT) was added to the reaction mixer. The samples were incubated at 40 ºC for 2 hours and then the RT and dsDNA synthesis was terminated by incubating at 70 ºC for 15min. The transcription master mix was prepared as the manufacturer’s protocol (4X Transcription buffer, 0.1M DTT, NTP mix, 50% PEG, RNase-Out, Inorganic pyrophosphatase, T7-RNA polymerase, and Cyanine 3/5-CTP). Transcription of dsDNA was performed by adding the transcription master mix to the dsDNA reaction samples and incubating at 40 oC for 2 hours. The transcirption reaction product was purified using the MEGAclear Kit (Amion, USA), according to the manufacturer’s instructions. Cyanine 3-labeled cRNA was fragmented by adding 10X blocking agent and 25X fragmentation buffer and incubating at 60 ºC for 30min.
Hybridization protocol
The fragmented cRNA of each samplte was resuspended with 2X hybridization buffer and directly pipetted onto each sub-array of the RoboArboChip v2 microarray. The arrays were hybridized at 65 ºC with a rotation of 10 rpm, for 17 h using the Agilent Hybridization oven (Agilent Technology, USA). The RoboArboChip slides were washed with the Gene Expression Wash Buffer 1 for 1min and then with the Gene Expression Wash Buffer 2 for 1min. The slides were added to a rack containing the Stabilization and Draying Solution for 30seconds.
Scan protocol
The RoboArboChip slides were scanned with the Axon GenePix 4000B scanner (Molecular Devices, USA) at a 532 nm wavelength with a pixel size of 10 μm.
Data processing
The median fluorescence intensity of each spot with local background subtraction were calculated from the scanned images with the GenePix Pro 7 software (Molecular Devices, USA). For data analysis, we have developed the RoboArboChip Analysis Form (RAF) using the Microsoft Excel software. The RAF contains an algorithm similar to that used by the DetectiV software (Watson et al., 2007). All the raw data were normalized against the negative control probes, distributed randomly in 78 spots of each sub-array; thus, the fluorescence intensity of each probe was divided by the mean fluorescence intensity of the negative control probes. After normalization, all the values £1 were transformed to 1. Normalized data were log2 transformed to reduce variability. The mean signal intensity of all the spots containing the probes of each virus species were calculated. The hypothesis that the mean signal intensity of the group of probes of each virus species is equal to the mean signal intensity of the negative control probes was tested by Welch’s t-test that is useful when two samples have unequal variances and unequal sample sizes, which is the case of the data obtained with the RoboArboChip platform. A virus was considered present in the 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 1 or higher than 1, i.e. at least two times higher than the mean signal intensity of the negative control probes.
