Whole blood RNA of uncomplicated dengue patient collected on day 4 of illness
Extracted molecule
total RNA
Extraction protocol
Whole-blood (2.5ml) was collected directly into PAXgene RNA tubes (Qiagen, Sussex, UK) at the bedside. RNA extraction was performed using Paxgene RNA kits (Qiagen). Purified RNA was stored at –80oC and quantified prior to use using a spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
Label
Streptavidine-Cy3
Label protocol
After hybridization, the chip was washed, blocked and stained with streptavidine-Cy3 for 10 min.
Hybridization protocol
Illumina hybridization standard procedure, in brief, Biotinylated amplified cRNA was generated by in vitro transcription (IVT) technology using Illumina® TotalPrep™ RNA Amplification Kit (Ambion, Inc., Austin, TX) according to the manufacturer’s instructions. After purification, 850ng of cRNA was hybridized to an Illumina HumanRef-8 V1BeadChip (containing probes to 23,961 RefSeq gene sequences) at 55 °C for 18 hours following the manufacturer’s instructions (Illumina, Inc., San Diego, CA).
Scan protocol
Chip was scanned using Illumina BeadArray Reader, scan factor 5
Description
RNA sample was first reverse transcribed to cDNA. the cDNA was then used to synthesis the antisense RNA using IVT protocol (Ambion, Inc., Austin, TX). The antisense RNA was then purified before 850ng of the product was used for hybridization
Data processing
Intensity was acquired using Beadstudio software Intensity was background normalised (Subtract the background value
