Total RNA was extracted with RNeasy Mini Kit followed by DNase I treatment.
Label
SYBR Green
Label protocol
PCR assays were performed using the Dendritic Cell - Antigen Presenting Cell and the Toll-like Receptor Pathway PCR assay panel (SA Biosciences, Frederick, MD) following the Manufacturer’s instructions. Reverse transcription was performed from 500 ng total RNA using the RT2 First Strand Kit (SA Biosciences). Quantitative real-time PCR were performed (Light Cycler 480, Roche) with 45 cycles at 94 oC for 15 seconds and 60 oC for 60 seconds.
Hybridization protocol
n/a
Scan protocol
n/a
Description
Test
Data processing
The normalization and all the data analysis were performed according to the manufacturers instructions using their web-based software package: http://sabiosciences.com/pcrarraydataanalysis.php For the normalization it uses the average of five housekeeping genes: B2M, HPRT1 , RPL13A, GAPDH, ACTB. Target gene signals normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ct_HKG)] The web-based software package automatically performs all deltadeltaCt based fold-change calculations from the uploaded raw threshold cycle data. Fold Change worksheet reports test/control ratios.
