Animals inoculated at either -5 or -3 days post-challenge (dpc) with 150ug/kg of PEGboIFN-lambda and/or 2.5x10^9 pfu Adt-O1M FMD vaccine. Animals were then challenged at 0dpc with 2x10^6 BID50 of FMDV O1Manisa
Extracted molecule
total RNA
Extraction protocol
PBMCs were isolated by applying heparinized blood to a ficoll gradient, and lysing with RLT lysis buffer. Total RNA was extracted using Qiagen Rneasy kits.
Label
FAM
Label protocol
First DNA is digested using a Sigma kit (Sigma, cat# D5307-1000UN), then cDNA is synthesized using Invitrogen reagents (Cat#: 28025-13, Y00147, 48190-001, 10777-019). Thermocycler Set to 25°C for 10min for 1 cycle, then 45 cycles of 37°C and 95°C for 5min before holding at 4°C indefinitely.
Hybridization protocol
n/a
Scan protocol
n/a
Data processing
Matrix normalized and Fold Change sheets generated in separate excel analysis worksheets and then copied here. All data normalized by subtracting average CT of both replicates of the gene of interest from the average CT of the housekeeping gene GAPDH. For ISGs CCL2 through IFNb, GAPDH reps 1 and 2 were used to normalize. For the adaptive immunity genes CD40L through CXCR4, GAPDH reps 3 and 4 were used for normalization. In instances where the baseline CT values were Undetermined after two attempts to run the qPCR, the average was taken of the CT values of the other biological replicates in that treatment group.
PEGboIFN-lambda as a FMD preexposure prophylactic and vaccine adjuvant in cattle—ISG and adaptive immunity gene expression
Data table header descriptions
ID_REF
VALUE
Normalized signal calculated in Microsoft Excel. Fold Change represents the comparison of the normalized gene expression level for a particular time point against the baseline measure for that gene in the same biological replicate (heifer).
