Fluorescent-labeled DNA (Cy3 and Cy5-dCTP) was produced through Eberwine’s linear RNA amplification method and subsequent enzymatic reaction. This procedure has been previously described in detail (Guo, Y. et al. J. Virol. 79, 14392-14403, 2005). Briefly, double-stranded cDNA containing T7 RNA polymerase promoter sequence was synthesized with 5 ug of total RNA using Reverse Transcription System, RNase H, DNA polymerase I and T4 DNA polymerase, according to the manufacturer’s recommended protocol (Promega).
Channel 2
Source name
MAQC sample A, i.e., Stratagene Universal Human Reference RNA (UHRR, Catalog #740000)
Fluorescent-labeled DNA (Cy3 and Cy5-dCTP) was produced through Eberwine’s linear RNA amplification method and subsequent enzymatic reaction. This procedure has been previously described in detail (Guo, Y. et al. J. Virol. 79, 14392-14403, 2005). Briefly, double-stranded cDNA containing T7 RNA polymerase promoter sequence was synthesized with 5 ug of total RNA using Reverse Transcription System, RNase H, DNA polymerase I and T4 DNA polymerase, according to the manufacturer’s recommended protocol (Promega).
Scan protocol
Slides were scanned with a confocal LuxScan scanner (CapitalBio Corp.). For two-color microarrays, the scanning setting for the Cy3 and Cy5 channels was manually balanced by visual inspection of the external control spots. The data from the obtained images were extracted with SpotData software (CapitalBio Corp.).
Description
Sample-pair convention: A (A/B, Cy3/Cy5), B (B/A, Cy3/Cy5), sA (A/A, Cy3/Cy5), sB (B/B, Cy3/Cy5). The human genome-wide long oligonucleotide microarray was constructed inhouse at CapitalBio Corporation, Beijing, China.
Data processing
The data from the obtained images were extracted with SpotData software (CapitalBio Corp.). The raw data were submitted to MAQC and further normalized by using linear-scaling and LOWESS.
