Total RNA was extracted with Qiagen RNeasy Mini Kit
Label
SYBR Green
Label protocol
Real Time qPCR was performed in a 25ml reaction mixture containing 12.5ml of RT2 SYBR green ROX qPCR master mix (catalog no: 330520), 1ml each of 10uM forward and reverse primers, 9.5ml of nuclease free water and 1ml of cDNA template, on the Qiagen Q5 plex thermal cycler. The PCR conditions were 94°C-2 min of initial denaturation followed by 35 cycles of 94°C-30 sec, 63°C-1 min, 72°C-1 min.
Hybridization protocol
n/a
Scan protocol
n/a
Description
control
Data processing
18srRNA was the house keeping gene used for normalization The statistical significance of the Relative expression ratios are taken from a paired T-test. For each subject the value of the expression ratio = log( Etarget )( Ctarget ) – log( Eref )( Cref) is computed. A paired t-test compares the mean of theses values between select subpopulations. The resulting test is the parametric equivalent of randomization tests implemented in the ‘REST’ PCR analysis software, where Relative expression ratio (R)= (E target) ΔCT target/(E ref) ΔCT ref where ΔCT target = Average control samples – Average experimental samples of the relevant target gene ΔCT ref = Average control samples – Average experimental samples of the reference house keeping gene Analysis is carried out with SAS version 9.4, SAS PROC MIXED.
