0.5 ug total RNA, 2.5 ul 1:1000 - 1:2^10 bacterial mRNA controls, 1ul T7 Oligo(dT) primer, add water to 12ul. Incubate for 10min at 70C. Add 2ul 10x First Strand Buffer, 4ul 5mM dNTP, 1ul RNase Inhibitor, 1ul ArrayScript to reaction. Incubate for 2h at 42C. Add 63ul water, 10ul 10x Second Starnd Buffer, 4ul 5mM dNTP Mix, 2ul DNA polymerase, 1ul RNaseH. Incubate for 2h at 16C. Purify the cNDA using column by adding 250ul cDNA Binding Buffer, spin at 10000g for 1min. Add 500ul Washing Buffer to column followed by spin at 10000g for 1 min. Spin dry the column. Add 12ul 50C water for disolving cDNA for 2min. Spin at 10000g for 1.5min. Repeat the elute step once. For labelling, add 12ul Biotin-NTP Mix, 4ul 10x T7 Reaction Buffer, 4ul 10x T7 Enzyme Mix to reaction. Incubate for 14h at 37C. Recover cRNA by columns. Add 60ul water and 350ul cRNA Binding Buffer to reaction. Transfer to column, spin and add 250ul 100% Ethanol and spin again. Add 650ul Washing Buffer, spin. Then spin dry the column. Add 100ul 50C water to disolve the cRNA.
Hybridization protocol
Fragment 10ug labeled cRNA in 20ul by adding 5ul 5x Fragmentation Buffer and incubate for 20C at 94C. Add 78ul BufferA, 130ul BufferB 27ul water and incubate for 5min at 90C to denature cRNA. Load reaction to bioarray chamber. Incubate for 22h at 37C on a 300rmp rotating platform. Wash slides using 0.75x TNT for 1hr at 46C. Stain slides using 2ug/ml Alexa Fluor 647-streptavidin in TNB buffer at room temprature for 30 min in dark. Then wash the slides 4 times using 1x TNT for 5min each time. Wasing slides using 0.1x SSC/ 0.05% Tw20 for 30sec. Dry slides at 2000rpm for 3min.
Scan protocol
Axon Genepix Pro 4100B scanner and GenePixPro 3.0 software, using wavelength 635nm, PTM 600V, 100% laser power, 5um pixel size, focus at 0um.
Description
Monocyte_HVL_020
Data processing
Discovery spots were loess normalized in R/BioConductor, then imported into GeneSpring GX 7.3.1 and normalized to 50th percentile per chip and median per gene, less than 0.01 set to 0.01.
