Total RNA was extracted with Qiagen RNeasy following the standard protocol, starting from ~10^6 cells
Label
SYBR Green
Label protocol
Reverse transcription was done with Life Technologies Vilo kit (standard protocol), starting from 150 ng total RNA. Quantitative real-time PCR was done in an 7900HT Real-Time PCR System (Life Technologies/ABI) using primer sets and SYBR Green from Qiagen, in 384-well plates, in a final volume of 5 μL and using default cycling parameters of the standard mode.
Hybridization protocol
n/a
Scan protocol
n/a
Description
Healthy subject
Data processing
We used Excel capabilities to calculate: (a) the average Cq from duplicate wells for each gene; (b) the median values of the three housekeeping genes (B2M, GAPDH and RPL13); (c) RCN as defined below. Detections were done in duplicate, and the values in Matrix non-normalized worksheet represent the average Cq of the duplicate per each sample. In the rare cases where one of the duplicates was Undetectable ot the dissociatin curve was not appropriate, we used the remaining sample. Sample data table reports the normalized signal [2^(-ΔCq)] against the median value of the three housekeeping genes: B2M, GAPDH and RPL13. In our analyses we used the Relative Copy Number [RCN: 2^(–ΔCq) × 100] (instead of calculating 'Fold change')
