array scanning protocol
normalization data transformation protocol
18s was used as an endogenous control for each gene expression assay. This allowed gene expression quantification for each cDNA sample relative to the 18s control and this is the data supplied in the Sample data table. We did not measure fold changes. Normalized CT values (ΔCT) for each amplified target gene replicate were calculated by taking the difference between the CT value of the target gene and its endogenous control. Resulting triplicate ΔCT values for individual target genes were averaged yielding a final ΔCT value. The 'raw data' prior to the delta Ct calculation were never provided by Source MDx. The 18s endogenous control was used as a quality control measure and all the delta Ct data supplied by Source MDx met the glp 18s quality control parameters (i.e., if the 'raw data' did not meet the 18s quality control, it would have been excluded by the quality control system from the delta Ct data sent to us). ID_REF = VALUE = Normalized CT values (ΔCT)
First strand cDNA was synthesized from random hexamer-primed RNA templates using TaqMan® Reverse Transcription reagents. Quantitative PCR (QPCR) analysis of the 18S rRNA content of newly synthesized cDNA, using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) served as a quality check of the first strand synthesis reaction. Target gene amplification was performed in a QPCR reaction using Applied Biosystem’s TaqMan® Universal PCR Master Mix and Source MDx® proprietary primer-probe sets (Precision Profiles™). Individual target gene amplification was multiplexed with the Eukaryotic 18S rRNA Endogenous Control and run in triplicate in a 384-well format on the 7900HT Fast Real-Time PCR System
nucleic acid extraction protocol
whole blood samples were collected in PAXgene Blood RNA tubes and were processed to total RNA within 30 days of phlebotomy with the Qiagen PAXgene Blood RNA kit.
sample treatment protocol